Process of preparing pharmaceutical compositions for the treatment of CFTR mediated diseases

ABSTRACT

Processes of preparing pharmaceutical compositions comprising 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl) cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1) in Form I and a solid dispersion comprising substantially amorphous N-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide (Compound 2), methods of treating, lessening the severity of or symptomatically treating CFTR mediated diseases, such as cystic fibrosis, methods of administering, and kits thereof are disclosed.

TECHNICAL FIELD OF INVENTION

The invention relates to a process of preparing pharmaceuticalcompositions comprising 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid (Compound 1)Form I and a solid dispersion comprising substantially amorphousN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(Compound 2), methods of treatment, methods of administering, and kitsthereof.

BACKGROUND

Cystic fibrosis (CF) is a recessive genetic disease that affectsapproximately 30,000 children and adults in the United States andapproximately 30,000 children and adults in Europe. Despite progress inthe treatment of CF, there is no cure.

In patients with CF, mutations in CFTR endogenously expressed inrespiratory epithelia leads to reduced apical anion secretion causing animbalance in ion and fluid transport. The resulting decrease in aniontransport contributes to enhanced mucus accumulation in the lung and theaccompanying microbial infections that ultimately cause death in CFpatients. In addition to respiratory disease, CF patients typicallysuffer from gastrointestinal problems and pancreatic insufficiency that,if left untreated, results in death. In addition, the majority of maleswith cystic fibrosis are infertile and fertility is decreased amongfemales with cystic fibrosis. In contrast to the severe effects of twocopies of the CF associated gene, individuals with a single copy of theCF associated gene exhibit increased resistance to cholera and todehydration resulting from diarrhea—perhaps explaining the relativelyhigh frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000 diseasecausing mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/app). The most prevalent mutationis a deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70% of the cases of cystic fibrosis and isassociated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Compound 1 in salt form is disclosed in International PCT PublicationWO2007056341 and U.S. Pat. No. 7,741,321 as an inducer of CFTR activityand thus as a useful treatment for CFTR-mediated diseases such as cysticfibrosis. Compound 1 Form I, which is a substantially crystalline andsalt-free form, is disclosed in International PCT PublicationWO2009073757 and U.S. Pat. No. 8,507,534. Compound 2 is disclosed inInternational PCT Publication WO2006002421 and U.S. Pat. No. 7,495,103as an inducer of CFTR activity and thus as useful treatment forCFTR-mediated diseases such as cystic fibrosis. A solid dispersioncomprising substantially amorphous Compound 2 is disclosed inInternational PCT Publication WO2010019239 and United States PublishedPatent Application No. US20100074949. All above applications and patentsare incorporated in their entirety by reference herein.

Compounds which are CFTR potentiators, such as Compound 2, and compoundswhich are CFTR correctors, such as Compound 1, have been shownindependently to have utility in the treatment of CFTR related diseases,such as cystic fibrosis.

Accordingly, there is a need for novel treatments of CFTR mediateddiseases which involve CFTR corrector and potentiator compounds.

Particularly, there is a need for combination therapies to treat CFTRmediated diseases, such as cystic fibrosis, which include CFTRpotentiator and corrector compounds.

More particularly, there is a need for combination therapies to treatCFTR mediated diseases, such as cystic fibrosis, which include CFTRpotentiator compounds, such as substantially amorphous Compound 2, incombination with CFTR corrector compounds, such as Compound 1 Form I.

Compound 1 as part of a combination with Compound 2 has been granted aBreakthrough Therapy Designation from the Food and Drug Administration(FDA) for the treatment of cystic fibrosis, one of only two such grantsat the time of the filing of this application (the other being forCompound 2). This demonstrates a significant unmet need for theeffective treatment of the cause of cystic fibrosis over symptomatictreatments. Additionally, a common challenge for drugs approved by theFDA is the occasional lack of drug availability for patients in needthereof. Accordingly, a significant unmet need exists for the presentlydisclosed Compound 1 and Compound 2 formulations and processes forpreparing them in a continuous and controlled manner.

Additionally, patient compliance with treatment schedules and dosageamounts is largely dependent on ease of drug administration. Apharmaceutical composition comprising fixed dosage amounts of a CFTRcorrector and CFTR potentiator, wherein the solid forms of saidcorrector and potentiator are stable, is a significant breakthrough forthe treatment of CFTR mediated diseases such as cystic fibrosis.

SUMMARY

The invention features a process of preparing pharmaceuticalcompositions comprising 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid, Compound 1Form I, which has the structure below:

anda solid dispersion of substantially amorphousN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide,Compound 2, which has the structure below:

methods of treatment, methods of administering, and kits thereof.

In one aspect, the present invention features a process of preparing apharmaceutical composition comprising:

a. Compound 1 Form I;

b. a solid dispersion comprising substantially amorphous Compound 2;

c. a filler;

d. a disintegrant;

e. a surfactant; and

f. a binder;

referred to as PC-I.

In one embodiment, the process of preparing pharmaceutical compositionsof the present invention comprise 30 to 55 percent by weight Compound 1Form I, and 10 to 45 percent by weight solid dispersion comprisingsubstantially amorphous Compound 2.

In one embodiment, the filler is selected from cellulose, modifiedcellulose, sodium carboxymethyl cellulose, ethyl cellulose hydroxymethylcellulose, hydroxypropylcellulose, cellulose acetate, microcrystallinecellulose, dibasic calcium phosphate, sucrose, lactose, corn starch,potato starch, or any combination thereof. In another embodiment, thefiller is microcrystalline cellulose, and is present in an amountranging from 10 to 20 percent by weight.

In one embodiment, the disintegrant is selected from agar-agar, algins,calcium carbonate, carboxymethylcellulose, cellulose,hydroxypropylcellulose, low substituted hydroxypropylcellulose, clays,croscarmellose sodium, crospovidone, gums, magnesium aluminum silicate,methylcellulose, polacrilin potassium, sodium alginate, sodium starchglycolate, maize starch, potato starch, tapioca starch, or anycombination thereof. In another embodiment, the disintegrant iscroscarmellose sodium, and is present in an amount ranging from 1 to 3percent by weight.

In one embodiment, the surfactant is selected from sodium laurylsulfate, sodium stearyl fumerate, polyoxyethylene 20 sorbitanmono-oleate, or any combination thereof. In another embodiment, thesurfactant is sodium lauryl sulfate, and is present in an amount rangingfrom 0.5 to 2 percent by weight.

In one embodiment, the binder is selected from polyvinylpyrrolidone,dibasic calcium phosphate, sucrose, corn starch, modified cellulose, orany combination thereof. In another embodiment, the binder ispolyvinylpyrrolidone, and is present in an amount ranging from 0 to 5percent by weight.

In one embodiment, the present invention features a process of preparinga pharmaceutical composition having the following formulation:

% by wgt. Compound 1 Form I 35-50 Solid dispersion comprisingsubstantially 25-40 amorphous Compound 2 Microcrystalline cellulose10-20 Croscarmellose sodium 1-3 Sodium lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5referred to as PC-II.

In another aspect, the present invention features a process of preparinga pharmaceutical composition comprising:

a. Compound 1 Form I;

b. a solid dispersion comprising substantially amorphous Compound 2;

c. a filler;

d. a disintegrant;

e. a surfactant;

f. a binder; and

g. a lubricant;

referred to as PC-III.

In one embodiment, the process of preparing pharmaceutical compositionsof present invention comprise about 100 to 250 mg of Compound 1 Form I,and about 100 to 150 mg of substantially amorphous Compound 2. Inanother embodiment, the pharmaceutical compositions of the presentinvention comprise about 200 mg of Compound 1 Form I, and about 125 mgof substantially amorphous Compound 2. In another embodiment, thepharmaceutical compositions of the present invention comprise about 150mg of Compound 1 Form I, and about 125 mg of substantially amorphousCompound 2.

In one embodiment, the process of preparing pharmaceutical compositionsof the present invention comprise 25 to 50 percent by weight Compound 1Form I, and 15 to 35 percent by weight a solid dispersion comprisingsubstantially amorphous Compound 2.

In one embodiment, the filler is selected from cellulose, modifiedcellulose, sodium carboxymethyl cellulose, ethyl cellulose hydroxymethylcellulose, hydroxypropylcellulose, cellulose acetate, microcrystallinecellulose, dibasic calcium phosphate, sucrose, lactose, corn starch,potato starch, or any combination thereof. In another embodiment, thefiller is microcrystalline cellulose, and is present in an amountranging from 20 to 30 percent by weight.

In one embodiment, the disintegrant is selected from agar-agar, algins,calcium carbonate, carboxymethylcellulose, cellulose,hydroxypropylcellulose, low substituted hydroxypropylcellulose, clays,croscarmellose sodium, crospovidone, gums, magnesium aluminum silicate,methylcellulose, polacrilin potassium, sodium alginate, sodium starchglycolate, maize starch, potato starch, tapioca starch, or anycombination thereof. In another embodiment, the disintegrant iscroscarmellose sodium, and is present in an amount ranging from 3 to 10percent by weight.

In one embodiment, the surfactant is selected from sodium laurylsulfate, sodium stearyl fumerate, polyoxyethylene 20 sorbitanmono-oleate, or any combination thereof. In another embodiment, thesurfactant is sodium lauryl sulfate, and is present in an amount rangingfrom 0.5 to 2 percent by weight.

In one embodiment, the binder is selected from polyvinylpyrrolidone,dibasic calcium phosphate, sucrose, corn starch, modified cellulose, orany combination thereof. In another embodiment, the binder ispolyvinylpyrrolidone, and is present in an amount ranging from 0 to 5percent by weight.

In one embodiment, the lubricant is selected from magnesium stearate,calcium stearate, zinc stearate, sodium stearate, stearic acid, aluminumstearate, leucine, glyceryl behenate, hydrogenated vegetable oil or anycombination thereof. In another embodiment, the lubricant is magnesiumstearate, and is present in an amount ranging from 0.5 to 2 percent byweight.

In one embodiment, the present invention features a process of preparinga pharmaceutical composition having the following formulation:

% by wgt. Compound 1 Form I 25-50 A solid dispersion comprisingsubstantially 15-35 amorphous Compound 2 Microcrystalline cellulose20-30 Croscarmellose sodium  3-10 Sodium lauryl sulfate 0.5-2 Polyvinylpyrrolidone 0-5 Magnesium stearate 0.5-2 referred to as PC-IV.

In one embodiment, the process of preparing pharmaceutical compositionsof the present invention further comprise a colorant and optionally awax. In another embodiment, the colorant is present in an amount rangingfrom 2 to 4 percent by weight. In another embodiment, the wax iscarnauba wax present in an amount ranging from 0 to 0.020 percent byweight.

In one embodiment, the process of preparing pharmaceutical compositionsof the present invention are solid oral pharmaceutical compositions. Inanother embodiment, the solid oral pharmaceutical compositions are agranular pharmaceutical composition or tablet.

In one embodiment, the process of preparing granular pharmaceuticalcompositions of the present invention have the following formulation:

% by wgt. Compound 1 Form I 43 Solid dispersion comprising substantially34 amorphous Compound 2 Microcrystalline cellulose 17 Croscarmellosesodium 2 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3referred to as PC-V.

In one embodiment, the process of preparing granular pharmaceuticalcompositions of the present invention have the following formulation:

% by wgt. Compound 1 Form I 38 Solid dispersion comprising substantially40 amorphous Compound 2 Microcrystalline cellulose 16 Croscarmellosesodium 2 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3referred to as PC-VI.

In one embodiment, the process of preparing granular pharmaceuticalcompositions of the present invention have the following formulation:

% by wgt. Compound 1 Form I 51 Solid dispersion comprising substantially27 amorphous Compound 2 Microcrystalline cellulose 16 Croscarmellosesodium 2 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3referred to as PC-VII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 35 Solid dispersion comprising substantially28 amorphous Compound 2 Microcrystalline cellulose 26 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1referred to as PC-VIII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 31 Solid dispersion comprising substantially32 amorphous Compound 2 Microcrystalline cellulose 26 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1referred to as PC-IX.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 41 Solid dispersion comprising substantially22 amorphous Compound 2 Microcrystalline cellulose 26 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1referred to as PC-X.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Solid dispersion comprising substantially 156amorphous Compound 2 Microcrystalline cellulose 150 Croscarmellosesodium 34 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 15 Magnesiumstearate 6referred to as PC-XI.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 150 Solid dispersion comprising substantially 156amorphous Compound 2 Microcrystalline cellulose 129 Croscarmellosesodium 30 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 13 Magnesiumstearate 5referred to as PC-XII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Solid dispersion comprising substantially 104amorphous Compound 2 Microcrystalline cellulose 128 Croscarmellosesodium 29 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 13 Magnesiumstearate 5referred to as PC-XIII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 34 Solid dispersion comprising substantially27 amorphous Compound 2 Microcrystalline cellulose 25 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1 Colorant 3referred to as PC-XIV.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 30 Solid dispersion comprising substantially31 amorphous Compound 2 Microcrystalline cellulose 25 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1 Colorant 3referred to as PC-XV.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

% by wgt. Compound 1 Form I 40 Solid dispersion comprising substantially21 amorphous Compound 2 Microcrystalline cellulose 25 Croscarmellosesodium 6 Sodium lauryl sulfate 1 Polyvinylpyrrolidone 3 Magnesiumstearate 1 Colorant 3referred to as PC-XVI.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Solid dispersion comprising substantially 156amorphous Compound 2 Microcrystalline cellulose 150 Croscarmellosesodium 34 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 15 Magnesiumstearate 6 Colorant 17referred to as PC-XVII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Substantially amorphous Compound 2 125Microcrystalline cellulose 150 Croscarmellose sodium 34 Sodium laurylsulfate 4 Polyvinylpyrrolidone 15 Magnesium stearate 6 Colorant 17referred to as PC-XVIII

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 150 Solid dispersion comprising substantially 156amorphous Compound 2 Microcrystalline cellulose 129 Croscarmellosesodium 29 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 13 Magnesiumstearate 5 Colorant 15referred to as PC-XIX.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Solid dispersion comprising substantially 104amorphous Compound 2 Microcrystalline cellulose 128 Croscarmellosesodium 29 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 13 Magnesiumstearate 5 Colorant 14referred to as PC-XX.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

mg Compound 1 Form I 200 Solid dispersion comprising substantially 83amorphous Compound 2 Microcrystalline cellulose 128 Croscarmellosesodium 29 Sodium lauryl sulfate 4 Polyvinylpyrrolidone 13 Magnesiumstearate 5 Colorant 14referred to as PC-XXI.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

Component % by wgt. Compound 1 Form I 20-40 Solid dispersion comprisingsubstantially 30-40 amorphous Compound 2 Microcrystalline cellulose20-30 Croscarmellose sodium  1-10 Polyvinylpyrrolidone 1-5 Sodium laurylsulfate 0.1-1  Magnesium stearate 0.5-1.5referred to as PC-XXII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

Compound 1/Compound 2 100 mg/125 mg Component % in Granule % in Tabletmg/Tablet Compound 1 Form I 30 25 100 Solid dispersion comprising 47 38156 substantially amorphous Compound 2 Microcrystalline cellulose 17 1355 Croscarmellose sodium 2 2 7 Polyvinylpyrrolidone 3 3 11 Sodium laurylsulfate 1 1 3 Total Granules 100 82 332 Croscarmellose sodium 4 18Microcrystalline cellulose 13 53 Magnesium stearate 1 4 Total Tablet 100407referred to as PC-XXIII.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

Compound 1/Compound 2 150 mg/125 mg Component % in Granule % in Tabletmg/Tablet Compound 1 Form I 38 31 150 Solid dispersion comprising 40 32156 substantially amorphous Compound 2 Microcrystalline cellulose 16 1365 Croscarmellose sodium 2 2 8 Polyvinylpyrrolidone 3 3 13 Sodium laurylsulfate 1 1 4 Total Granules 100 82 396 Croscarmellose sodium 4 22Microcrystalline cellulose 13 64 Magnesium stearate 1 5 Total Tablet 100487referred to as PC-XXIV.

In one embodiment, the process of preparing tablets of the presentinvention have the following formulation:

Compound 1/Compound 2 75 mg/125 mg Component % in Granule % in Tabletmg/Tablet Compound 1 Form I 25 20 75 Solid dispersion comprising 52 43156 substantially amorphous Compound 2 Microcrystalline cellulose 17 1349 Croscarmellose sodium 2 2 6 Polyvinylpyrrolidone 3 3 10 Sodium laurylsulfate 1 1 3 Total Granules 100 82 299 Croscarmellose sodium 4 17Microcrystalline cellulose 13 48 Magnesium stearate 1 4 Core Tablet 100368 Pink Opadry 3 11 Film Coated Tablet 379Referred to as PC-XXV.

In one aspect, the present invention features a method of treating,lessening the severity of, or symptomatically treating cystic fibrosisin a patient comprising administering to the patient an effective amountof the pharmaceutical composition, granular pharmaceutical composition,or tablet of the present invention.

In embodiment, the present invention features a method of treating,lessening the severity of, or symptomatically treating cystic fibrosisin a patient comprising administering to the patient an effective amountof the pharmaceutical composition, granular pharmaceutical composition,or tablet of any one of formulations PC-I through PC-XXV.

In one embodiment, the patient has a ΔF508 CFTR mutation. In anotherembodiment, the patient is homozygous in ΔF508. In another embodiment,the patient is heterozygous in ΔF508. In another embodiment, two tabletsare administered to the patient per day.

In one aspect, the present invention features a method of preparing agranular pharmaceutical composition comprising wet granulating thefollowing components:

a. Compound 1 Form I;

b. a solid dispersion comprising substantially amorphous Compound 2;

c. a filler;

d. a disintegrant;

e. a surfactant; and

f. a binder.

In one aspect the present invention features a method of preparing atablet comprising compressing:

i) a plurality of granular pharmaceutical compositions comprising thefollowing components:

-   -   a. Compound 1 Form I;    -   b. a solid dispersion comprising substantially amorphous        Compound 2;    -   c. a filler;    -   d. a disintegrant;    -   e. a surfactant; and    -   f. a binder;

ii) a disintegrant;

iii) a filler; and

iv) a lubricant.

In one aspect, the present invention features a kit comprisingpharmaceutical compositions, granular pharmaceutical compositions, ortablets of the present invention, and a separate therapeutic agent orpharmaceutical composition thereof.

In one embodiment, the pharmaceutical compositions, granularpharmaceutical compositions, or tablets of the present invention, andthe separate therapeutic agent or pharmaceutical composition thereof arein separate containers. In another embodiment, the separate containersare bottles. In another embodiment, the separate containers are vials.In another embodiment, the separate containers are blister packs.

In another aspect, the invention provides a continuous orsemi-continuous process for making the pharmaceutical compositionsdescribed herein by a twin screw wet granulation process comprising thesteps of screening and weighing Compound 1, Compound 2, and excipients;mixing Compound 1, Compound 2, and excipients in a blender and feedingthe blend into a continuous granulator while adding a granulation fluidcomprising surfactant and a binder at a suitable rate for a suitableamount of time and chopping the mixture into granules; drying thegranules; blending the granules with extra-granular excipients for asuitable amount of time; compressing the blend into tablets; coating thetablets; and, optionally, printing a monogram on one or both tabletfaces.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an X-ray diffraction pattern calculated from a single crystalstructure of Compound 1 Form I.

FIG. 2 is an actual X-ray powder diffraction pattern of Compound 1 FormI.

FIG. 3 is a graph depicting Compound 1 pH gradient dissolution profilesfor a tablet made by a high shear granulation (HSG) process and a twinscrew wet granulation (TSWG) process (LOD stands for loss on drying, ameasure to define the amount of water in a powder/granule).

FIG. 4 is a graph depicting the stability of the substantially amorphousform of Compound 2 in tablet formulation PC-XVII at 50° C. afterpre-equilibrating at 60% relative humidity by showing only a smallamount of crystallinity over time.

FIG. 5 is a graph depicting the stability of the substantially amorphousform of Compound 2 in tablet formulation PC-XVII at 60° C. afterpre-equilibrating at 60% relative humidity by showing only a smallamount of crystallinity over time.

FIG. 6 is a graph depicting the stability of the substantially amorphousform of Compound 2 in tablet formulation PC-XX at 60° C. afterpre-equilibrating at 60% relative humidity by showing only a smallamount of crystallinity over time.

FIG. 7 is a graph depicting the stability of the substantially amorphousform of Compound 2 in tablet formulation PC-XX at 50° C. afterpre-equilibrating at 60% relative humidity by showing only a smallamount of crystallinity over time.

FIG. 8 is an ¹HNMR spectrum of Compound 1.

FIG. 9 is an ¹HNMR spectrum of Compound 1 HCl salt.

FIG. 10 is a differential scanning calorimetry (DSC) trace of Compound 1Form I.

FIG. 11 is a conformational picture of Compound 1 Form I based on singlecrystal X-ray analysis.

FIG. 12 is a schematic drawing of a process analytical technique (PAT)enabled continuous manufacturing process where in step 1) feeder/blenderone, PAT1 NIR measures material attributes during screening of rawmaterials; step 2) twin screw granulator, PAT2 NIR measures compositionand BU; step 3) fluidized bed dryer, PAT 3a NIR measures granuleuniformity, LOD, solid state form and physical attributes of granules,PAT 3b laser diffraction measures particle size distribution; step 4)milling, PAT4 NIR measures composition and BU; step 5) feeder/blendertwo, PAT 5a Raman measures assay and CU, PAT 5b weight, hardness,thickness; step 6) compression, PAT6 Raman measures coat thickness; andstep 7) coating.

FIG. 13 is a schematic drawing showing a PAT inline Sentronics NIRlocated after blender one, granule mill, and extra granule blender. Eachprobe has 7 spots that cycle sequentially to maximize sampling and NIRwith multiplexer-NIR ensuring robust and exhaustive sampling bycontrolled powder flow across the probe optics.

FIG. 14 is a depiction of NIR in flowing powder.

FIG. 15 is a Kaiser Raman spectrum of Compound 1 Form I and Compound 1Form II (Compound 1 Form II is a different polymorph disclosed in US201131588 incorporated herein in its entirety by reference) taken aftertablet pressing. The Kaiser Raman spectrometer is mounted on the KraemerUTS tablet tester.

FIG. 16 is a graph showing good correlation between predicted andreference off-line NIR samplings of Compound 2 granules.

FIG. 17 is a series of NIR spectra measuring water content in samples ofCompound 1 granules.

FIG. 18 is a series of NIR spectra measuring a range of compositionscomprising different ratios of Compound 1 Form I and a solid dispersionscomprising substantially amorphous Compound 2 on the left, andpretreated spectra on the right depicting Range A for identifyingCompound 1 Form I and Range B for identifying amorphous Compound 2.

FIG. 19 depicts a calibration curve for predicted Compound 1 Form Icontent versus reference (actual) Compound 1 Form I content usingpartial least squares (PLS) techniques.

FIG. 20 depicts actual results of unknown samples comprising differentcontents of Compound 1 Form I (Y Reference) versus predicted contentusing the calibration curve calculated from FIG. 19 (Y Predicted).

FIG. 21 depicts the transmission percent of a laser diffractionmeasurement in response to changes in line rate (flow velocity) for acomposition comprising Compound 1 Form I and a solid dispersionscomprising substantially amorphous Compound 2 showing the expectedreduction in transmission percent as line rate increase.

FIG. 22 depicts laser diffraction measurements of particles comprisingCompound 1 Form I and a solid dispersions comprising substantiallyamorphous Compound 2 at different line rates showing that the averageparticle size (Dv(50) is not affected by line rate.

FIG. 23 depicts laser diffraction measurements of particles comprisingCompound 1 Form I and a solid dispersions comprising substantiallyamorphous Compound 2 under different processing parameters showing thatthe particle size measurements are sensitive to such changes.

FIG. 24 depicts the predictive capabilities of process analyticaltechnology models using Raman spectroscopy, both non-continuously andcontinuously, for monitoring Compound 1 solid form identity in a tablet.

FIG. 25 depicts the predictive capabilities of process analyticaltechnology models using Raman spectroscopy, both non-continuously andcontinuously, for monitoring Compound 2 solid form identity in a tablet.

FIG. 26 depicts a flow chart, which describes a process for continuouslypreparing a tablet comprising Compound 1 and Compound 2.

DETAILED DESCRIPTION Definitions

As used herein, “CFTR” stands for cystic fibrosis transmembraneconductance regulator.

As used herein, a “ΔF508 mutation” or “F508-del mutation” is a specificmutation within the CFTR protein. The mutation is a deletion of thethree nucleotides that comprise the codon for amino acid phenylalanineat position 508, resulting in CFTR protein that lacks this phenylalanineresidue.

As used herein, a patient who is “homozygous” for a particular mutation,e.g. ΔF508, has the same mutation on each allele.

As used herein, a patient who is “heterozygous” for a particularmutation, e.g. ΔF508, has this mutation on one allele, and a differentmutation on the other allele.

As used herein, the term “CFTR corrector” refers to a compound thatincreases the amount of functional CFTR protein to the cell surface,resulting in enhanced ion transport.

As used herein, the term “CFTR potentiator” refers to a compound thatincreases the channel activity of CFTR protein located at the cellsurface, resulting in enhanced ion transport.

As used herein, the term “active pharmaceutical ingredient” or “API”refers to a biologically active compound.

As used herein, the term “PAT” stands for process analytical technology.

As used herein, the term “CU” stands for content uniformity.

The terms “solid form”, “solid forms” and related terms, when usedherein refer to Compound 1 or Compound 2, in a particular solid forme.g. crystals, amorphous states, and the like.

As used herein, the term “substantially amorphous” refers to a solidmaterial having little or no long range order in the position of itsmolecules. For example, substantially amorphous materials have less thanabout 15% crystallinity (e.g., less than about 10% crystallinity or lessthan about 5% crystallinity). It is also noted that the term‘substantially amorphous’ includes the descriptor, ‘amorphous’, whichrefers to materials having no (0%) crystallinity.

As used herein, the term “substantially crystalline” (as in the phrasesubstantially crystalline Compound 1 Form I refers to a solid materialhaving predominantly long range order in the position of its molecules.For example, substantially crystalline materials have more than about85% crystallinity (e.g., more than about 90% crystallinity or more thanabout 95% crystallinity). It is also noted that the term ‘substantiallycrystalline’ includes the descriptor, ‘crystalline’, which refers tomaterials having 100% crystallinity.

The term “crystalline” and related terms used herein, when used todescribe a substance, component, product, or form, means that thesubstance, component or product is substantially crystalline asdetermined by X-ray diffraction. (See, e.g., Remington: The Science andPractice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins,Baltimore, Md. (2003); The United States Pharmacopeia, 23^(rd) ed.,1843-1844 (1995)).

As used herein, an “excipient” includes functional and non-functionalingredients in a pharmaceutical composition.

As used herein, a “disintegrant” is an excipient that hydrates apharmaceutical composition and aids in tablet dispersion. As usedherein, a “diluent” or “filler” is an excipient that adds bulkiness to apharmaceutical composition.

As used herein, a “surfactant” is an excipient that impartspharmaceutical compositions with enhanced solubility and/or wettability.

As used herein, a “binder” is an excipient that imparts a pharmaceuticalcomposition with enhanced cohesion or tensile strength (e.g., hardness).

As used herein, a “glidant” is an excipient that imparts apharmaceutical compositions with enhanced flow properties.

As used herein, a “colorant” is an excipient that imparts apharmaceutical composition, e.g. a tablet, with a desired color.Examples of colorants include commercially available pigments such asFD&C Blue #1 Aluminum Lake, FD&C Blue #2, other FD&C Blue colors,titanium dioxide, iron oxide, and/or combinations thereof. In oneembodiment, the tablet provided by the invention is pink.

As used herein, a “lubricant” is an excipient that is added topharmaceutical compositions that are pressed into tablets. The lubricantaids in compaction of granules into tablets and ejection of a tablet ofa pharmaceutical composition from a die press.

As used herein, “cubic centimeter” and “cc” are used interchangeably torepresent a unit of volume. Note that 1 cc=1 mL.

As used herein, “kiloPond” and “kP” are used interchangeably and referto the measure of force where a kP=approximately 9.8 Newtons.

As used herein, “friability” refers to the property of a tablet toremain intact and hold its form despite an external force of pressure.Friability can be quantified using the mathematical expression presentedin equation 1:

$\begin{matrix}{{\%\mspace{14mu}{friability}} = {100 \times \frac{\left( {W_{0} - W_{f}} \right)}{W_{0}}}} & (1)\end{matrix}$wherein W₀ is the original weight of the tablet and W is the finalweight of the tablet after it is put through the friabilator. Friabilityis measured using a standard USP testing apparatus that tumblesexperimental tablets for 100 or 400 revolutions. Some tablets of theinvention have a friability of less than 5.0%. In another embodiment,the friability is less than 2.0%. In another embodiment, the targetfriability is less than 1.0% after 400 revolutions.

As used herein, “mean particle diameter” Is the average particlediameter as measured using techniques such as laser light scattering,image analysis, or sieve analysis. In one embodiment, the granules usedto prepare the pharmaceutical compositions provided by the inventionhave a mean particle diameter of less than 1.0 mm.

As used herein, “bulk density” is the mass of particles of materialdivided by the total volume the particles occupy. The total volumeincludes particle volume, inter-particle void volume and internal porevolume. Bulk density is not an intrinsic property of a material; it canchange depending on how the material is processed. In one embodiment,the granules used to prepare the pharmaceutical compositions provided bythe invention have a bulk density of about 0.5-0.7 g/cc.

An “effective amount” or “therapeutically effective amount” of acompound of the invention may vary according to factors such as thedisease state, age, and weight of the subject, and the ability of thecompound of the invention to elicit a desired response in the subject.Dosage regimens may be adjusted to provide the optimum therapeuticresponse. An effective amount is also one in which any toxic ordetrimental effects (e.g., side effects) of the compound of theinvention are outweighed by the therapeutically beneficial effects.

As used herein, and unless otherwise specified, the terms“therapeutically effective amount” and “effective amount” of a compoundmean an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or disorder, or to delay orminimize one or more symptoms associated with the disease or disorder. A“therapeutically effective amount” and “effective amount” of a compoundmean an amount of therapeutic agent, alone or in combination with one ormore other agent(s), which provides a therapeutic benefit in thetreatment or management of the disease or disorder. The terms“therapeutically effective amount” and “effective amount” can encompassan amount that improves overall therapy, reduces or avoids symptoms orcauses of disease or disorder, or enhances the therapeutic efficacy ofanother therapeutic agent.

“Substantially pure” as used in the phrase “substantially pure Compound1 Form I” means greater than about 90% purity. In another embodiment,substantially pure refers to greater than about 95% purity. In anotherembodiment, substantially pure refers to greater than about 98% purity.In another embodiment, substantially pure refers to greater than about99% purity.

With respect to Compound 1 Form I, or a solid dispersion comprisingsubstantially amorphous Compound 2, the terms “about” and“approximately”, when used in connection with doses, amounts, or weightpercent of ingredients of a composition or a dosage form, mean a dose,amount, or weight percent that is recognized by one of ordinary skill inthe art to provide a pharmacological effect equivalent to that obtainedfrom the specified dose, amount, or weight percent. Specifically theterm “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or 0.05% of a given value or range.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising Compound 1Form I and a solid dispersion comprising substantially amorphousCompound 2. In some embodiments of this aspect, the amount of Compound 1Form I that is present in the pharmaceutical composition is 100 mg, 125mg, 150 mg, 200 mg, 250 mg, 300 mg, or 400 mg. In some embodiments ofthis aspect, wgt. percent of Compound 1 Form I present in thepharmaceutical composition is from 10 to 75 percent. In these and otherembodiments, Compound 1 Form I is present as substantially pure Compound1 Form I. In some embodiments of this aspect, the amount ofsubstantially amorphous Compound 2 that is present in the pharmaceuticalcomposition is 100 mg, 125 mg, 150 mg, 200 mg, or 250 mg. In someembodiments of this aspect, wgt. percent of substantially amorphousCompound 2 that is present in the pharmaceutical composition is from 10to 75 percent. In these and other embodiments, substantially amorphousCompound 2 is present as substantially pure and amorphous Compound 2.“Substantially pure” means greater than ninety percent pure; preferablygreater than 95 percent pure; more preferably greater than 99.5 percentpure.

Thus in one aspect, the invention provides a pharmaceutical compositioncomprising:

a. Compound 1 Form I;

b. a solid dispersion of substantially amorphous Compound 2;

c. a filler;

d. a disintegrant;

e. a surfactant; and

f. a binder.

In one embodiment of this aspect, the pharmaceutical compositioncomprises 25 mg of Compound 1 Form I. In another embodiment of thisaspect, the pharmaceutical composition comprises 50 mg of Compound 1Form I. In another embodiment of this aspect, the pharmaceuticalcomposition comprises 100 mg of Compound 1 Form I. In another embodimentof this aspect, the pharmaceutical composition comprises 125 mg ofCompound 1 Form I. In another embodiment of this aspect, thepharmaceutical composition comprises 150 mg of Compound 1 Form I. Inanother embodiment of this aspect, the pharmaceutical compositioncomprises 200 mg of Compound 1 Form I. In another embodiment of thisaspect, the pharmaceutical composition comprises 250 mg of Compound 1Form I. In another embodiment of this aspect, the pharmaceuticalcomposition comprises 400 mg of Compound 1 Form I.

In one embodiment of this aspect, the pharmaceutical compositioncomprises 25 mg of substantially amorphous Compound 2. In anotherembodiment of this aspect, the pharmaceutical composition comprises 50mg of substantially amorphous Compound 2. In another embodiment of thisaspect, the pharmaceutical composition comprises 100 mg of substantiallyamorphous Compound 2. In another embodiment of this aspect, thepharmaceutical composition comprises 125 mg of substantially amorphousCompound 2. In another embodiment of this aspect, the pharmaceuticalcomposition comprises 150 mg of substantially amorphous Compound 2. Inanother embodiment of this aspect, the pharmaceutical compositioncomprises 200 mg of substantially amorphous Compound 2. In anotherembodiment of this aspect, the pharmaceutical composition comprises 250mg of substantially amorphous Compound 2.

In some embodiments, the pharmaceutical compositions comprises Compound1 Form I, wherein Compound 1 Form I is present in an amount of at least15 wt % (e.g., at least 20 wt %, at least 30 wt %, at least 40 wt %, atleast 50 wt %, or at least 60 wt %) by weight of the composition.

In some embodiments, the pharmaceutical compositions comprisessubstantially amorphous Compound 2, wherein the substantially amorphousCompound 2 is present in an amount of at least 15 wt % (e.g., at least20 wt %, at least 30 wt %, at least 40 wt %, at least 50 wt %, or atleast 60 wt %) by weight of the composition.

In some embodiments, the pharmaceutical composition comprises Compound 1Form I, a solid dispersion comprising substantially amorphous Compound2, a filler, a disintegrant, a surfactant, and a binder. In thisembodiment, the composition comprises from about 25 wt % to about 55 wt% (e.g., about 30-50 wt %) of Compound 1 Form I by weight of thecomposition, and more typically, from 40 wt % to about 45 wt % ofCompound 1 Form I by weight of the composition. In this embodiment, thecomposition comprises from about 15 wt % to about 40 wt % (e.g., about20-35 wt %) of substantially amorphous Compound 2 by weight of thecomposition, and more typically, from 25 wt % to about 30 wt % ofsubstantially amorphous Compound 2 by weight of the composition.

The concentration of Compound 1 Form I and substantially amorphousCompound 2 in the composition depends on several factors such as theamount of pharmaceutical composition needed to provide a desired amountof Compound 1 Form I and substantially amorphous Compound 2 and thedesired dissolution profile of the pharmaceutical composition.

In another embodiment, the pharmaceutical composition comprises Compound1 Form I, in which Compound 1 Form I in its solid form has a meanparticle diameter, measured by light scattering (e.g., using a MalvernMastersizer available from Malvern Instruments in England) of 0.1microns to 10 microns. In another embodiment, the particle size ofCompound 1 Form I is 1 micron to 5 microns. In another embodiment,Compound 1 Form I has a particle size D50 of 2.0 microns.

As indicated, in addition to Compound 1 Form I and a solid dispersion ofsubstantially amorphous Compound 2, in some embodiments of theinvention, the pharmaceutical compositions which are oral formulationsalso comprise one or more excipients such as fillers, disintegrants,surfactants, diluents, binders, glidants, lubricants, colorants, orfragrances and any combination thereof.

Fillers suitable for the invention are compatible with the ingredientsof the pharmaceutical composition, i.e., they do not substantiallyreduce the solubility, the hardness, the chemical stability, thephysical stability, or the biological activity of the pharmaceuticalcomposition. Exemplary fillers include: celluloses, modified celluloses,(e.g. sodium carboxymethyl cellulose, ethyl cellulose hydroxymethylcellulose, hydroxypropylcellulose), cellulose acetate, microcrystallinecellulose, calcium phosphates, dibasic calcium phosphate, starches (e.g.corn starch, potato starch), sugars (e.g., sorbitol) lactose, sucrose,or the like), or any combination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises atleast one filler in an amount of at least 5 wt % (e.g., at least about20 wt %, at least about 30 wt %, or at least about 40 wt %) by weight ofthe composition. For example, the pharmaceutical composition comprisesfrom about 10 wt % to about 60 wt % (e.g., from about 20 wt % to about55 wt %, from about 25 wt % to about 50 wt %, or from about 27 wt % toabout 45 wt %) of filler, by weight of the composition. In anotherexample, the pharmaceutical composition comprises at least about 20 wt %(e.g., at least 30 wt % or at least 40 wt %) of microcrystallinecellulose, for example MCC Avicel PH102, by weight of the composition.In yet another example, the pharmaceutical composition comprises fromabout 10 wt % to about 60 wt % (e.g., from about 20 wt % to about 55 wt% or from about 25 wt % to about 45 wt %) of microcellulose, by weightof the composition.

Disintegrants suitable for the invention enhance the dispersal of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, the hardness, or thebiological activity of the pharmaceutical composition. Exemplarydisintegrants include croscarmellose sodium, sodium starch glycolate, ora combination thereof.

Thus, in one embodiment, the pharmaceutical composition comprisesdisintegrant in an amount of about 10 wt % or less (e.g., about 7 wt %or less, about 6 wt % or less, or about 5 wt % or less) by weight of thecomposition. For example, the pharmaceutical composition comprises fromabout 1 wt % to about 10 wt % (e.g., from about 1.5 wt % to about 7.5 wt% or from about 2.5 wt % to about 6 wt %) of disintegrant, by weight ofthe composition. In another example, the pharmaceutical compositioncomprises about 10 wt % or less (e.g., 7 wt % or less, 6 wt % or less,or 5 wt % or less) of croscarmellose sodium, by weight of thecomposition. In yet another example, the pharmaceutical compositioncomprises from about 1 wt % to about 10 wt % (e.g., from about 1.5 wt %to about 7.5 wt % or from about 2.5 wt % to about 6 wt %) ofcroscarmellose sodium, by weight of the composition. In some examples,the pharmaceutical composition comprises from about 0.1% to about 10 wt% (e.g., from about 0.5 wt % to about 7.5 wt % or from about 1.5 wt % toabout 6 wt %) of disintegrant, by weight of the composition. In stillother examples, the pharmaceutical composition comprises from about 0.5%to about 10 wt % (e.g., from about 1.5 wt % to about 7.5 wt % or fromabout 2.5 wt % to about 6 wt %) of disintegrant, by weight of thecomposition.

Surfactants suitable for the invention enhance the wettability of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, the hardness, or thebiological activity of the pharmaceutical composition. Exemplarysurfactants include sodium lauryl sulfate (SLS), sodium stearyl fumarate(SSF), polyoxyethylene 20 sorbitan mono-oleate (e.g., Tween™), anycombination thereof, or the like.

Thus, in one embodiment, the pharmaceutical composition comprises asurfactant in an amount of about 10 wt % or less (e.g., about 5 wt % orless, about 2 wt % or less, about 1 wt % or less, about 0.8 wt % orless, or about 0.6 wt % or less) by weight of the composition. Forexample, the pharmaceutical composition includes from about 10 wt % toabout 0.1 wt % (e.g., from about 5 wt % to about 0.2 wt % or from about2 wt % to about 0.3 wt %) of surfactant, by weight of the composition.In another example, the pharmaceutical composition comprises 10 wt % orless (e.g., about 5 wt % or less, about 2 wt % or less, about 1 wt % orless, about 0.8 wt % or less, or about 0.6 wt % or less) of sodiumlauryl sulfate, by weight of the composition. In yet another example,the pharmaceutical composition comprises from about 10 wt % to about 0.1wt % (e.g., from about 5 wt % to about 0.2 wt % or from about 2 wt % toabout 0.3 wt %) of sodium lauryl sulfate, by weight of the composition.

Binders suitable for the invention enhance the tablet strength of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe chemical stability, the physical stability, or the biologicalactivity of the pharmaceutical composition. Exemplary binders includepolyvinylpyrrolidone, dibasic calcium phosphate, sucrose, corn (maize)starch, modified cellulose (e.g., hydroxymethyl cellulose), or anycombination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises abinder in an amount of at least about 0.1 wt % (e.g., at least about 1wt %, at least about 3 wt %, at least about 4 wt %, or at least about 5wt %) by weight of the composition. For example, the pharmaceuticalcomposition comprises from about 0.1 wt % to about 10 wt % (e.g., fromabout 1 wt % to about 10 wt % or from about 2 wt % to about 7 wt %) ofbinder, by weight of the composition. In another example, thepharmaceutical composition comprises at least about 0.1 wt % (e.g., atleast about 1 wt %, at least about 2 wt %, at least about 3 wt %, or atleast about 4 wt %) of polyvinylpyrrolidone, by weight of thecomposition. In yet another example, the pharmaceutical compositioncomprises a glidant in an amount ranging from about 0.1 wt % to about 10wt % (e.g., from about 1 wt % to about 8 wt % or from about 2 wt % toabout 5 wt %) of polyvinylpyrrolidone, by weight of the composition.

Diluents suitable for the invention may add necessary bulk to aformulation to prepare tablets of the desired size and are generallycompatible with the ingredients of the pharmaceutical composition, i.e.,they do not substantially reduce the solubility, the hardness, thechemical stability, the physical stability, or the biological activityof the pharmaceutical composition. Exemplary diluents include: sugars,for example, confectioner's sugar, compressible sugar, dextrates,dextrin, dextrose, lactose, mannitol, sorbitol, cellulose, and modifiedcelluloses, for example, powdered cellulose, talc, calcium phosphate,starch, or any combination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises adiluent in an amount of 40 wt % or less (e.g., 35 wt % or less, 30 wt %or less, or 25 wt % or less, or 20 wt % or less, or 15 wt % or less, or10 wt % or less) by weight of the composition. For example, thepharmaceutical composition comprises from about 40 wt % to about 1 wt %(e.g., from about 35 wt % to about 5 wt % or from about 30 wt % to about7 wt %, from about 25 wt % to about 10 wt %, from about 20 wt % to about15 wt %) of diluent, by weight of the composition. In another example,the pharmaceutical composition comprises 40 wt % or less (e.g., 35 wt %or less, 25 wt % or less, or 15 wt % or less) of mannitol, by weight ofthe composition. In yet another example, the pharmaceutical compositioncomprises from about 35 wt % to about 1 wt % (e.g., from about 30 wt %to about 5 wt % or from about 25 wt % to about 10 wt %) of mannitol, byweight of the composition.

Glidants suitable for the invention enhance the flow properties of thepharmaceutical composition and are compatible with the ingredients ofthe pharmaceutical composition, i.e., they do not substantially reducethe solubility, the hardness, the chemical stability, the physicalstability, or the biological activity of the pharmaceutical composition.Exemplary glidants include colloidal silicon dioxide, talc, or acombination thereof.

Thus, in one embodiment, the pharmaceutical composition comprises aglidant in an amount of 2 wt % or less (e.g., 1.75 wt %, 1.25 wt % orless, or 1.00 wt % or less) by weight of the composition. For example,the pharmaceutical composition comprises from about 2 wt % to about 0.05wt % (e.g., from about 1.5 wt % to about 0.07 wt % or from about 1.0 wt% to about 0.09 wt %) of glidant, by weight of the composition. Inanother example, the pharmaceutical composition comprises 2 wt % or less(e.g., 1.75 wt %, 1.25 wt % or less, or 1.00 wt % or less) of colloidalsilicon dioxide, by weight of the composition. In yet another example,the pharmaceutical composition comprises from about 2 wt % to about 0.05wt % (e.g., from about 1.5 wt % to about 0.07 wt % or from about 1.0 wt% to about 0.09 wt %) of colloidal silicon dioxide, by weight of thecomposition.

In some embodiments, the pharmaceutical composition can include an oralsolid pharmaceutical dosage form which can comprise a lubricant that canprevent adhesion of a granulate-bead admixture to a surface (e.g., asurface of a mixing bowl, a compression die and/or punch). A lubricantcan also reduce interparticle friction within the granulate and improvethe compression and ejection of compressed pharmaceutical compositionsfrom a die press. The lubricant is also compatible with the ingredientsof the pharmaceutical composition, i.e., they do not substantiallyreduce the solubility, the hardness, or the biological activity of thepharmaceutical composition. Exemplary lubricants include magnesiumstearate, calcium stearate, zinc stearate, sodium stearate, stearicacid, aluminum stearate, leucine, glyceryl behenate, hydrogenatedvegetable oil or any combination thereof. In one embodiment, thepharmaceutical composition comprises a lubricant in an amount of 5 wt %or less (e.g., 4.75 wt %, 4.0 wt % or less, or 3.00 wt % or less, or 2.0wt % or less) by weight of the composition. For example, thepharmaceutical composition comprises from about 5 wt % to about 0.10 wt% (e.g., from about 4.5 wt % to about 0.5 wt % or from about 3 wt % toabout 1 wt %) of lubricant, by weight of the composition. In anotherexample, the pharmaceutical composition comprises 5 wt % or less (e.g.,4.0 wt % or less, 3.0 wt % or less, or 2.0 wt % or less, or 1.0 wt % orless) of magnesium stearate, by weight of the composition. In yetanother example, the pharmaceutical composition comprises from about 5wt % to about 0.10 wt % (e.g., from about 4.5 wt % to about 0.15 wt % orfrom about 3.0 wt % to about 0.50 wt %) of magnesium stearate, by weightof the composition.

Pharmaceutical compositions of the invention can optionally comprise oneor more colorants, flavors, and/or fragrances to enhance the visualappeal, taste, and/or scent of the composition. Suitable colorants,flavors, or fragrances are compatible with the ingredients of thepharmaceutical composition, i.e., they do not substantially reduce thesolubility, the chemical stability, the physical stability, thehardness, or the biological activity of the pharmaceutical composition.In one embodiment, the pharmaceutical composition comprises a colorant,a flavor, and/or a fragrance. In one embodiment, the pharmaceuticalcompositions provided by the invention are purple.

In some embodiments, the pharmaceutical composition includes or can bemade into tablets and the tablets can be coated with a colorant andoptionally labeled with a logo, other image and/or text using a suitableink. In still other embodiments, the pharmaceutical composition includesor can be made into tablets and the tablets can be coated with acolorant, waxed, and optionally labeled with a logo, other image and/ortext using a suitable ink. Suitable colorants and inks are compatiblewith the ingredients of the pharmaceutical composition, i.e., they donot substantially reduce the solubility, the chemical stability, thephysical stability, the hardness, or the biological activity of thepharmaceutical composition. The suitable colorants and inks can be anycolor and are water based or solvent based. In one embodiment, tabletsmade from the pharmaceutical composition are coated with a colorant andthen labeled with a logo, other image, and/or text using a suitable ink.For example, tablets comprising pharmaceutical composition as describedherein can be coated with about 3 wt % (e.g., less than about 6 wt % orless than about 4 wt %) of film coating comprising a colorant. Thecolored tablets can be labeled with a logo and text indicating thestrength of the active ingredient in the tablet using a suitable ink. Inanother example, tablets comprising pharmaceutical composition asdescribed herein can be coated with about 3 wt % (e.g., less than about6 wt % or less than about 4 wt %) of a film coating comprising acolorant.

In another embodiment, tablets made from the pharmaceutical compositionare coated with a colorant, waxed, and then labeled with a logo, otherimage, and/or text using a suitable ink. For example, tablets comprisingpharmaceutical composition as described herein can be coated with about3 wt % (e.g., less than about 6 wt % or less than about 4 wt %) of filmcoating comprising a colorant. The colored tablets can be waxed withCarnauba wax powder weighed out in the amount of about 0.01% w/w of thestarting tablet core weight. The waxed tablets can be labeled with alogo and text indicating the strength of the active ingredient in thetablet using a suitable ink. In another example, tablets comprisingpharmaceutical composition as described herein can be coated with about3 wt % (e.g., less than about 6 wt % or less than about 4 wt %) of afilm coating comprising a colorant The colored tablets can be waxed withCarnauba wax powder weighed out in the amount of about 0.01% w/w of thestarting tablet core weight. The waxed tablets can be labeled with alogo and text indicating the strength of the active ingredient in thetablet using a pharmaceutical grade ink such as a black ink (e.g.,Opacode® S-1-17823, a solvent based ink, commercially available fromColorcon, Inc. of West Point, Pa.).

One exemplary pharmaceutical composition comprises from about 15 wt % toabout 70 wt % (e.g., from about 15 wt % to about 60 wt %, from about 15wt % to about 50 wt %, or from about 20 wt % to about 70 wt %, or fromabout 30 wt % to about 70 wt %) of Compound 1 Form I, by weight of thecomposition; and from about 15 wt % to about 40 wt % (e.g., about 20-35wt %) of substantially amorphous Compound 2 by weight of thecomposition, and more typically, from 25 wt % to about 30 wt % ofsubstantially amorphous Compound 2 by weight of the composition. Theaforementioned compositions can also include one or morepharmaceutically acceptable excipients, for example, from about 20 wt %to about 50 wt % of a filler; from about 1 wt % to about 5 wt % of adisintegrant; from about 2 wt % to about 0.3 wt % of a surfactant; andfrom about 0.1 wt % to about 5 wt % of a binder.

Another exemplary pharmaceutical composition comprises from about 15 wt% to about 70 wt % (e.g., from about 15 wt % to about 60 wt %, fromabout 15 wt % to about 50 wt %, or from about 15 wt % to about 40 wt %or from about 20 wt % to about 70 wt %, or from about 30 wt % to about70 wt %, or from about 40 wt % to about 70 wt %, or from about 50 wt %to about 70 wt %) of Compound 1 Form I by weight of the composition,from about 15 wt % to about 40 wt % (e.g., about 20-35 wt %) ofsubstantially amorphous Compound 2 by weight of the composition, andmore typically, from 25 wt % to about 30 wt % of substantially amorphousCompound 2 by weight of the composition, and one or more excipients, forexample, from about 20 wt % to about 50 wt % of a filler; from about 1wt % to about 5 wt % of a disintegrant; from about 2 wt % to about 0.3wt % of a surfactant; from about 0.1 wt % to about 5 wt % of a binder;and from about 2 wt % to about 0.1 wt % of a lubricant.

Another exemplary pharmaceutical composition comprises from about 15 wt% to about 70 wt % (e.g., from about 15 wt % to about 60 wt %, fromabout 15 wt % to about 50 wt %, or from about 15 wt % to about 40 wt %or from about 20 wt % to about 70 wt %, or from about 30 wt % to about70 wt %, or from about 40 wt % to about 70 wt %, or from about 50 wt %to about 70 wt %) of Compound 1 Form I by weight of the composition,from about 15 wt % to about 40 wt % (e.g., about 20-35 wt %) ofsubstantially amorphous Compound 2 by weight of the composition, andmore typically, from 25 wt % to about 30 wt % of substantially amorphousCompound 2 by weight of the composition, and one or more excipients, forexample, from about 20 wt % to about 50 wt % of a filler; from about 1wt % to about 5 wt % of a disintegrant; from about 2 wt % to about 0.3wt % of a surfactant; from about 0.1 wt % to about 5 wt % of a binder;from about 2 wt % to about 0.1 wt % of a lubricant; from about 2 wt % toabout 4 wt % colorant; and about 0.005 wt % to about 0.015 wt % wax.

In one embodiment, the invention is a granular pharmaceuticalcomposition comprising:

a. about 43 wt % of Compound 1 Form I by weight of the composition;

b. about 34 wt % of a solid dispersion comprising substantiallyamorphous Compound 2 by weight of the composition;

c. about 17 wt % of microcrystalline cellulose by weight of thecomposition;

d. about 2 wt % of croscarmellose sodium by weight of the composition;

e. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

f. about 3 wt % of polyvinylpyrrolidone by weight of the composition.

In one embodiment, the invention is a tablet comprising:

a. about 35 wt % of Compound 1 Form I by weight of the composition;

b. about 28 wt %/o of a solid dispersion comprising substantiallyamorphous Compound 2 by weight of the composition;

c. about 26 wt % of microcrystalline cellulose by weight of thecomposition;

d. about 6 wt % of croscarmellose sodium by weight of the composition;

e. about 3 wt % of polyvinylpyrrolidone by weight of the composition;

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

g. about 1 wt % of magnesium stearate by weight of the composition.

In one embodiment, the invention is a tablet comprising:

a. about 34 wt % of Compound 1 Form I by weight of the composition;

b. about 27 wt % of a solid dispersion comprising substantiallyamorphous Compound 2 by weight of the composition;

c. about 26 wt % of microcrystalline cellulose by weight of thecomposition;

d. about 6 wt % of croscarmellose sodium by weight of the composition;

e. about 2 wt % of polyvinylpyrrolidone by weight of the composition

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;

g. about 1 wt % of magnesium stearate by weight of the composition;

h. about 3 wt % of a colorant by weight of the composition; and

i. about 0.010 wt % of a wax by weight of the composition.

Another tablet of the invention comprises:

a. about 150 to 250 mg of Compound 1 Form I;

b. about 100 to 150 mg of substantially amorphous Compound 2;

c. about 125 to 175 mg of microcrystalline cellulose;

d. about 20 to 40 mg of croscarmellose sodium;

e. about 10 to 20 mg of polyvinylpyrrolidone;

f. about 2 to 6 mg of sodium lauryl sulfate; and

g. about 3 to 7 mg of magnesium stearate.

Another tablet of the invention comprises:

a. about 200 mg of Compound 1 Form I;

b. about 125 mg of substantially amorphous Compound 2;

c. about 150 mg of microcrystalline cellulose;

d. about 34 mg of croscarmellose sodium;

e. about 15 mg of polyvinylpyrrolidone;

f. about 4 mg of sodium lauryl sulfate; and

g. about 6 mg of magnesium stearate.

Another tablet of the invention comprises:

a. about 200 mg of Compound 1 Form I;

b. about 125 mg of substantially amorphous Compound 2;

c. about 150 mg of microcrystalline cellulose;

d. about 34 mg of croscarmellose sodium;

e. about 15 mg of polyvinylpyrrolidone;

f. about 4 mg of sodium lauryl sulfate;

g. about 6 mg of magnesium stearate;

h. about 17 mg of a colorant; and

i. about 0.06 mg of a wax.

In one embodiment, the invention is a granular pharmaceuticalcomposition comprising:

a. about 38 wt % of Compound 1 Form I by weight of the composition;

b. about 40 wt % of a solid dispersion comprising substantiallyamorphous Compound 2 by weight of the composition;

c. about 16 wt % of microcrystalline cellulose by weight of thecomposition;

d. about 2 wt % of croscarmellose sodium by weight of the composition;

e. about 1 wt % of sodium lauryl sulfate by weight of the composition;and

f. about 3 wt % of polyvinylpyrrolidone by weight of the composition.

In one embodiment, the invention is a tablet comprising:

a. about 31 wt % of Compound 1 Form I by weight of the composition;

b. about 32 wt % of a solid dispersion comprising substantiallyamorphous Compound 2 by weight of the composition;

c. about 26 wt % of microcrystalline cellulose by weight of thecomposition;

d. about 6 wt % of croscarmellose sodium by weight of the composition;

e. about 3 wt % of polyvinylpyrrolidone by weight of the composition

f. about 1 wt % of sodium lauryl sulfate by weight of the composition;

g. about 1 wt % of magnesium stearate by weight of the composition; and

h. about 3 wt % of a colorant by weight of the composition.

Another tablet of the invention comprises:

a. about 100 to 200 mg of Compound 1 Form I;

b. about 100 to 150 mg of substantially amorphous Compound 2;

c. about 100 to 150 mg of microcrystalline cellulose;

d. about 20 to 40 mg of croscarmellose sodium;

e. about 10 to 20 mg of polyvinylpyrrolidone;

f. about 2 to 6 mg of sodium lauryl sulfate; and

g. about 3 to 7 mg of magnesium stearate.

Another tablet of the invention comprises:

a. about 150 mg of Compound 1 Form I;

b. about 125 mg of substantially amorphous Compound 2;

c. about 129 mg of microcrystalline cellulose;

d. about 29 mg of croscarmellose sodium;

e. about 13 mg of polyvinylpyrrolidone;

f. about 4 mg of sodium lauryl sulfate;

g. about 5 mg of magnesium stearate; and

h. about 15 mg of a colorant.

The pharmaceutical compositions of the invention can be processed into atablet form, capsule form, pouch form, lozenge form, or other solid formthat is suited for oral administration. Thus in some embodiments, thepharmaceutical compositions are in tablet form.

Another aspect of the invention provides a pharmaceutical formulationconsisting of a tablet that includes Compound 1 Form I, a soliddispersion comprising substantially amorphous Compound 2, and excipients(e.g., a filler, a disintegrant, a surfactant, a binder, a colorant, alubricant, or any combination thereof), each of which is described aboveand in the Examples below, wherein the tablet has a dissolution of atleast about 50% (e.g., at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, or at least about 99%) in about 30minutes.

In one example, the pharmaceutical composition consists of a tablet thatincludes Compound 1 Form I in an amount ranging from 25 mg to 400 mg,for example, 25 mg, or 50 mg, or 75 mg, or 100 mg, or 150 mg, 200 mg,250 mg, 300 mg, or 400 mg, substantially amorphous Compound 2 in anamount ranging from 25 mg to 250 mg, for example, 25 mg, or 50 mg, or 75mg, or 100 mg, or 150 mg, 200 mg, 250 mg, and one or more excipients(e.g., a filler, a disintegrant, a surfactant, a binder, a colorant, alubricant, or any combination thereof) each of which is described aboveand in the Examples below, wherein the tablet has a dissolution of fromabout 50% to about 100% (e.g., from about 55% to about 95% or from about60% to about 90%) in about 30 minutes.

Dissolution can be measured with a standard USP Type II apparatus thatemploys a dissolution media of 0.1% CTAB dissolved in 900 mL of DIwater, buffered at pH 6.8 with 50 mM potassium phosphate monoasic,stirring at about 50-75 rpm at a temperature of about 37° C. A singleexperimental tablet is tested in each test vessel of the apparatus.Dissolution can also be measured with a standard USP Type II apparatusthat employs a dissolution media of 0.7% sodium lauryl sulfate dissolvedin 900 mL of 50 mM sodium phosphate buffer (pH 6.8), stirring at about65 rpm at a temperature of about 37° C. A single experimental tablet istested in each test vessel of the apparatus. Dissolution can also bemeasured with a standard USP Type II apparatus that employs adissolution media of 0.5% sodium lauryl sulfate dissolved in 900 mL of50 mM sodium phosphate buffer (pH 6.8), stirring at about 65 rpm at atemperature of about 37° C. A single experimental tablet is tested ineach test vessel of the apparatus.

Methods for Making Compound 1 Form I and a Solid Dispersion ComprisingSubstantially Amorphous Compound 2

Compound 1

Compound 1 is used as the starting point for Compound 1 Form I and canbe prepared by coupling an acid chloride moiety with an amine moietyaccording to Schemes 1-4.

Scheme 1 depicts the preparation of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride,which is used in Scheme 3 to make the amide linkage of Compound 1.

The starting material, 2,2-difluorobenzo[d][1,3]dioxole-5-carboxylicacid, is commercially available from Saltigo (an affiliate of theLanxess Corporation). Reduction of the carboxylic acid moiety in2,2-difluorobenzo[d][1,3]dioxole-5-carboxylic acid to the primaryalcohol, followed by conversion to the corresponding chloride usingthionyl chloride (SOCl₂), provides5-(chloromethyl)-2,2-difluorobenzo[d][1,3]dioxole, which is subsequentlyconverted to 2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile usingsodium cyanide. Treatment of2-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)acetonitrile with base and1-bromo-2-chloroethane provides1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile. Thenitrile moiety in1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonitrile isconverted to a carboxylic acid using base to give1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxylic acid,which is converted to the desired acid chloride using thionyl chloride.

Scheme 2 depicts an alternative synthesis of the requisite acidchloride. 5-bromomethyl-2,2-difluoro-1,3-benzodioxole is coupled withethyl cyanoacetate in the presence of a palladium catalyst to form thecorresponding alpha cyano ethyl ester. Saponification of the estermoiety to the carboxylic acid gives the cyanoethyl compound. Alkylationof the cyanoethyl compound with 1-bromo-2-chloro ethane in the presenceof base gives the cyanocyclopropyl compound. Treatment of thecyanocyclopropyl compound with base gives the carboxylate salt, which isconverted to the carboxylic acid by treatment with acid. Conversion ofthe carboxylic acid to the acid chloride is then accomplished using achlorinating agent such as thionyl chloride or the like.

Scheme 3 depicts the preparation of the requisite tert-butyl3-(6-amino-3-methylpyridin-2-yl)benzoate, which is coupled with1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloride inScheme 3 to give Compound 1. Palladium-catalyzed coupling of2-bromo-3-methylpyridine with 3-(tert-butoxycarbonyl)phenylboronic acidgives tert-butyl 3-(3-methylpyridin-2-yl)benzoate, which is subsequentlyconverted to the desired compound.

Scheme 4 depicts the coupling of1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarbonyl chloridewith tert-butyl 3-(6-amino-3-methylpyridin-2-yl)benzoate using triethylamine and 4-dimethylaminopyridine to initially provide the tert-butylester of Compound 1.

Compound 1 Form I

Compound 1 Form I is prepared by dispersing or dissolving a salt form,such as the HCl salt, of Compound 1 in an appropriate solvent for aneffective amount of time. Treatment of the tert-butyl ester with an acidsuch as HCl, gives the HCL salt of Compound 1, which is typically acrystalline solid. Compound 1 Form I may also be prepared directly fromthe t-butyl ester precursor by treatment with an appropriate acid, suchas formic acid.

The HCl salt of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid can be usedto make Form I by dispersing or dissolving the HCl salt of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid in anappropriate solvent for an effective amount of time. Other salts of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid may be used,such as, for example, salts derived from other mineral or organic acids.The other salts result from acid-mediated hydrolysis of the t-butylester moiety. Salts derived from other acids may include, for example,nitric, sulfuric, phosphoric, boric, acetic, benzoic and malonic. Thesesalt forms of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid may or maynot be soluble, depending upon the solvent used, but lack of solubilitydoes not hinder formation of Compound 1 Form I. For example, in oneembodiment, the appropriate solvent may be water or an alcohol/watermixture such as 50% methanol/water mixture, even though the HCl saltform of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is onlysparingly soluble in water. In one embodiment, the appropriate solventis water.

The effective amount of time for formation of Compound 1 Form I from thesalt of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid can be anytime between 2 to 24 hours or greater. It is recognized that the amountof time needed is inversely proportional to the temperature. That is,the higher the temperature the less time needed to affect dissociationof acid to form Compound 1 Form I. When the solvent is water, stirringthe dispersion for approximately 24 hours at room temperature providesCompound 1 Form I in an approximately 98% yield. If a solution of thesalt of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid is desiredfor process purposes, an elevated temperature may be used. Afterstirring the solution for an effective amount of time at the elevatedtemperature, recrystallization upon cooling provides substantially pureCompound 1 Form I. In one embodiment, substantially pure refers togreater than about 90% purity. In another embodiment, substantially purerefers to greater than about 95% purity. In another embodiment,substantially pure refers to greater than about 98% purity. In anotherembodiment, substantially pure refers to greater than about 99% purity.The temperature selected depends in part on the solvent used and is wellwithin the determination capabilities of one of ordinary skill in theart. In one embodiment, the temperature is between room temperature andabout 80° C. In another embodiment, the temperature is between roomtemperature and about 40° C. In another embodiment, the temperature isbetween about 40° C. and about 60° C. In another embodiment, thetemperature is between about 60° C. and about 80° C.

Compound 1 Form I may also be formed directly from3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (cf.Scheme 3), which is a precursor to the salt of Compound 1. Thus,3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate isallowed to undergo reaction with an appropriate acid, such as, forexample, formic acid under appropriate reaction conditions to giveCompound 1 Form I.

Compound 1 Form I may be further purified by recrystallization from anorganic solvent. Examples of organic solvents include, but are notlimited to, toluene, cumene, anisol, 1-butanol, isopropyl acetate, butylacetate, isobutyl acetate, methyl t-butyl ether, methyl isobutyl ketoneand 1-propanol-water mixtures. The temperature may be as describedabove. For example, Compound 1 Form I is dissolved in I-butanol at 75°C. until it is completely dissolved. Cooling down the solution to 10° C.at a rate of 0.2° C./min yields crystals of Compound 1 Form I which maybe isolated by filtration.

In one embodiment, Compound 1 Form I is characterized by one or morepeaks at 15.2 to 15.6 degrees, 16.1 to 16.5 degrees, and 14.3 to 14.7degrees in an X-ray powder diffraction obtained using Cu K alpharadiation. In another embodiment, Compound 1 Form I is characterized byone or more peaks at 15.4, 16.3, and 14.5 degrees. In anotherembodiment, Compound 1 Form I is further characterized by a peak at 14.6to 15.0 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 14.8 degrees. In another embodiment, Compound1 Form I is further characterized by a peak at 17.6 to 18.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 17.8 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 16.4 to 16.8 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 16.4 to 16.8degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 16.6 degrees. In another embodiment, Compound1 Form I is further characterized by a peak at 7.6 to 8.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 7.8 degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 25.8 to 26.2 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 26.0 degrees. Inanother embodiment, Compound 1 Form I is further characterized by a peakat 21.4 to 21.8 degrees. In another embodiment, Compound 1 Form I isfurther characterized by a peak at 21.6 degrees. In another embodiment,Compound 1 Form I is further characterized by a peak at 23.1 to 23.5degrees. In another embodiment, Compound 1 Form I is furthercharacterized by a peak at 23.3 degrees. In some embodiments, Compound 1Form I is characterized by a diffraction pattern substantially similarto that of FIG. 1. In some embodiments, Compound 1 Form I ischaracterized by a diffraction pattern substantially similar to that ofFIG. 2.

In some embodiments, the particle size distribution of D90 is about 82μm or less for Compound 1 Form I. In some embodiments, the particle sizedistribution of D50 is about 30 μm or less for Compound 1 Form I.

Compound 2

Compound 2 is the starting point for the solid dispersion comprisingsubstantially amorphous Compound 2 and can be prepared by coupling a4-oxo-dihydroquinoline carboxylic acid moiety with an amine moietyaccording to Schemes 5-7.

Solid Dispersion Comprising Substantially Amorphous Compound 2

Starting from Compound 2 the amorphous form of Compound 2 may beprepared by spray dried methods. Spray drying is a process that convertsa liquid feed to a dried particulate form. Optionally, a secondarydrying process such as fluidized bed drying or vacuum drying, may beused to reduce residual solvents to pharmaceutically acceptable levels.Typically, spray drying involves contacting a highly dispersed liquidsuspension or solution, and a sufficient volume of hot air to produceevaporation and drying of the liquid droplets. The preparation to bespray dried can be any solution, coarse suspension, slurry, colloidaldispersion, or paste that may be atomized using the selected spraydrying apparatus. In a standard procedure, the preparation is sprayedinto a current of warm filtered air that evaporates the solvent andconveys the dried product to a collector (e.g. a cyclone). The spent airis then exhausted with the solvent, or alternatively the spent air issent to a condenser to capture and potentially recycle the solvent.Commercially available types of apparatus may be used to conduct thespray drying. For example, commercial spray dryers are manufactured byBuchi Ltd. And Niro (e.g., the PSD line of spray driers manufactured byNiro) (see, US 2004/0105820; US 2003/0144257).

Spray drying typically employs solid loads of material from about 3% toabout 30% by weight, (i.e., drug and excipients), for example about 4%to about 20% by weight, preferably at least about 10%. In general, theupper limit of solid loads is governed by the viscosity of (e.g., theability to pump) the resulting solution and the solubility of thecomponents in the solution. Generally, the viscosity of the solution candetermine the size of the particle in the resulting powder product.

Techniques and methods for spray drying may be found in Perry's ChemicalEngineering Handbook, 6^(th) Ed., R. H. Perry, D. W. Green & J. O.Maloney, eds.), McGraw-Hill book co. (1984); and Marshall “Atomizationand Spray-Drying” 50, Chem. Eng. Prog. Monogr. Series 2 (1954). Ingeneral, the spray drying is conducted with an inlet temperature of fromabout 60° C. to about 200° C., for example, from about 95° C. to about185° C., from about 110° C. to about 182° C., from about 96° C. to about180° C., e.g., about 145° C. The spray drying is generally conductedwith an outlet temperature of from about 30° C. to about 90° C., forexample from about 40° C. to about 80° C., about 45° C. to about 80° C.e.g., about 75° C. The atomization flow rate is generally from about 4kg/h to about 12 kg/h, for example, from about 4.3 kg/h to about 10.5kg/h, e.g., about 6 kg/h or about 10.5 kg/h. The feed flow rate isgenerally from about 3 kg/h to about 10 kg/h, for example, from about3.5 kg/h to about 9.0 kg/h, e.g., about 8 kg/h or about 7.1 kg/h. Theatomization ratio is generally from about 0.3 to 1.7, e.g., from about0.5 to 1.5, e.g., about 0.8 or about 1.5.

Removal of the solvent may require a subsequent drying step, such astray drying, fluid bed drying (e.g., from about room temperature toabout 100° C.), vacuum drying, microwave drying, rotary drum drying orbiconical vacuum drying (e.g., from about room temperature to about 200°C.).

In one embodiment, the spray dried dispersion is fluid bed dried.

In one process, the solvent includes a volatile solvent, for example asolvent having a boiling point of less than about 100° C. In someembodiments, the solvent includes a mixture of solvents, for example amixture of volatile solvents or a mixture of volatile and non-volatilesolvents. Where mixtures of solvents are used, the mixture can includeone or more non-volatile solvents, for example, where the non-volatilesolvent is present in the mixture at less than about 15%, e.g., lessthan about 12%, less than about 10%, less than about 8%, less than about5%, less than about 3%, or less than about 2%.

Preferred solvents are those solvents where Compound 2 has a solubilityof at least about 10 mg/ml, (e.g., at least about 15 mg/ml, 20 mg/ml, 25mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, or greater).More preferred solvents include those where Compound 2 has a solubilityof at least about 20 mg/ml.

Exemplary solvents that could be tested include acetone, cyclohexane,dichloromethane, N,N-dimethylacetamide (DMA), N,N-dimethylformamide(DMF), 1,3-dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide (DMSO),dioxane, ethyl acetate, ethyl ether, glacial acetic acid (HAc), methylethyl ketone (MEK), N-methyl-2-pyrrolidinone (NMP), methyl tert-butylether (MTBE), tetrahydrofuran (THF), pentane, acetonitrile, methanol,ethanol, isopropyl alcohol, isopropyl acetate, and toluene. Exemplaryco-solvents include acetone/DMSO, acetone/DMF, acetone/water, MEK/water,THF/water, dioxane/water. In a two solvent system, the solvents can bepresent in of from about 0.1% to about 99.9%. In some preferredembodiments, water is a co-solvent with acetone where water is presentfrom about 0.1% to about 15%, for example about 9% to about 11%, e.g.,about 10%. In some preferred embodiments, water is a co-solvent with MEKwhere water is present from about 0.1% to about 15%, for example about9% to about 11%, e.g., about 10%. In some embodiments the solventsolution include three solvents. For example, acetone and water can bemixed with a third solvent such as DMA, DMF, DMI, DMSO, or HAc. Ininstances where substantially amorphous Compound 2 is a component of asolid dispersion, preferred solvents dissolve both Compound 2 and thepolymer. Suitable solvents include those described above, for example,MEK, acetone, water, methanol, and mixtures thereof.

The particle size and the temperature drying range may be modified toprepare an optimal spray dry dispersion. As would be appreciated byskilled practitioners, a small particle size would lead to improvedsolvent removal. Applicants have found however, that smaller particlescan lead to fluffy particles that, under some circumstances do notprovide optimal spray dry dispersions for downstream processing such astableting. At higher temperatures, crystallization or chemicaldegradation of substantially amorphous Compound 2 may occur. At lowertemperatures, a sufficient amount of the solvent may not be removed. Themethods herein provide an optimal particle size and an optimal dryingtemperature.

In general, particle size is such that D10 (μm) is less than about 5,e.g., less than about 4.5, less than about 4.0, or less than about 3.5,D50 (μm) is generally less than about 17, e.g., less than about 16, lessthan about 15, less than about 14, less than about 13, and D90 (μm) isgenerally less than about 175, e.g., less than about 170, less thanabout 170, less than about 150, less than about 125, less than about100, less than about 90, less than about 80, less than about 70, lessthan about 60, or less than about less than about 50. In general bulkdensity of the spray dried particles is from about 0.08 g/cc to about0.20 g/cc, e.g., from about 0.10 to about 0.15 g/cc, e.g., about 0.11g/cc or about 0.14 g/cc. Tap density of the spray dried particlesgenerally ranges from about 0.08 g/cc to about 0.20 g/cc, e.g., fromabout 0.10 to about 0.15 g/cc, e.g., about 0.11 g/cc or about 0.14 g/cc,for 10 taps; 0.10 g/cc to about 0.25 g/cc, e.g., from about 0.11 toabout 0.21 g/cc, e.g., about 0.15 g/cc, about 0.19 g/cc, or about 0.21g/cc for 500 taps; 0.15 g/cc to about 0.27 g/cc, e.g., from about 0.18to about 0.24 g/cc, e.g., about 0.18 g/cc, about 0.19 g/cc, about 0.20g/cc, or about 0.24 g/cc for 1250 taps; and 0.15 g/cc to about 0.27g/cc, e.g., from about 0.18 to about 0.24 g/cc, e.g., about 0.18 g/cc,about 0.21 g/cc, about 0.23 g/cc, or about 0.24 g/cc for 2500 taps.

Polymers

Spray dried dispersions including amorphous Compound 2 and a polymer (orsolid state carrier) also are included herein. For example, Compound 2is present as an amorphous compound as a component of a solid amorphousdispersion. The solid amorphous dispersion, generally includessubstantially amorphous Compound 2 and a polymer. Exemplary polymersinclude cellulosic polymers such as HPMC or HPMCAS and pyrrolidonecontaining polymers such as PVP/VA. In some embodiments, the solidamporphous dispersion includes one or more additional excipients, suchas a surfactant.

In one embodiment, a polymer is able to dissolve in aqueous media. Thesolubility of the polymers may be pH-independent or pH-dependent. Thelatter include one or more enteric polymers. The term “enteric polymer”refers to a polymer that is preferentially soluble in the less acidicenvironment of the intestine relative to the more acid environment ofthe stomach, for example, a polymer that is insoluble in acidic aqueousmedia but soluble when the pH is above 5-6. An appropriate polymershould be chemically and biologically inert. In order to improve thephysical stability of the spray dry dispersions, the glass transitiontemperature (T) of the polymer should be as high as possible. Forexample, preferred polymers have a glass transition temperature at leastequal to or greater than the glass transition temperature of the drug(i.e., Compound 2). Other preferred polymers have a glass transitiontemperature that is within about 10 to about 15° C. of the drug (i.e.,Compound 2). Examples of suitable glass transition temperatures of thepolymers include at least about 90° C., at least about 95° C., at leastabout 100° C., at least about 105° C., at least about 110° C., at leastabout 115° C., at least about 120° C., at least about 125° C., at leastabout 130° C., at least about 135° C., at least about 140° C., at leastabout 145° C., at least about 150° C., at least about 155° C., at leastabout 160° C., at least about 165° C., at least about 170° C., or atleast about 175° C. (as measured under dry conditions). Without wishingto be bound by theory, it is believed that the underlying mechanism isthat a polymer with a higher T_(g) generally has lower molecularmobility at room temperature, which can be a crucial factor instabilizing the physical stability of the amorphous spray drydispersion.

Additionally, the hygroscopicity of the polymers should be as low, e.g.,less than about 10%. For the purpose of comparison in this application,the hygroscopicity of a polymer or composition is characterized at about60% relative humidity. In some preferred embodiments, the polymer hasless than about 10% water absorption, for example less than about 9%,less than about 8%, less than about 7%, less than about 6%, less thanabout 5%, less than about 4%, less than about 3%, or less than about 2%water absorption. The hygroscopicity can also affect the physicalstability of the spray dry dispersions. Generally, moisture adsorbed inthe polymers can greatly reduce the T_(g) of the polymers as well as theresulting spray dry dispersions, which will further reduce the physicalstability of the spray dry dispersions as described above.

In one embodiment, the polymer is one or more water-soluble polymer(s)or partially water-soluble polymer(s). Water-soluble or partiallywater-soluble polymers include but are not limited to, cellulosederivatives (e.g., hydroxypropylmethylcellulose (HPMC),hydroxypropylcellulose (HPC)) or ethylcellulose; polyvinylpyrrolidones(PVP); polyethylene glycols (PEG); polyvinyl alcohols (PVA); acrylates,such as polymethacrylate (e.g., Eudragit® E); cyclodextrins (e.g.,β-cyclodextin) and copolymers and derivatives thereof, including forexample PVP-VA (polyvinylpyrollidone-vinyl acetate).

In some embodiments, the polymer is hydroxypropylmethylcellulose (HPMC),such as HPMCAS, HPMC E50, HPMCE15, or HPMC60SH50).

As discussed herein, the polymer can be a pH-dependent enteric polymer.Such pH-dependent enteric polymers include, but are not limited to,cellulose derivatives (e.g., cellulose acetate phthalate (CAP)),hydroxypropyl methyl cellulose phthalates (HPMCP), hydroxypropyl methylcellulose acetate succinate (HPMCAS), carboxymethylcellulose (CMC) or asalt thereof (e.g., a sodium salt such as (CMC-Na)); cellulose acetatetrimellitate (CAT), hydroxypropylcellulose acetate phthalate (HPCAP),hydroxypropylmethyl-cellulose acetate phthalate (HPMCAP), andmethylcellulose acetate phthalate (MCAP), or polymethacrylates (e.g.,Eudragit® S). In some embodiments, the polymer is hydroxypropyl methylcellulose acetate succinate (HPMCAS). In some embodiments, the polymeris hydroxypropyl methyl cellulose acetate succinate HG grade(HPMCAS-HG).

In yet another embodiment, the polymer is a polyvinylpyrrolidoneco-polymer, for example, a vinylpyrrolidone/vinyl acetate co-polymer(PVP/VA).

In embodiments where Compound 2 forms a spray dry dispersion with apolymer, for example with an HPMC, HPMCAS, or PVP/VA polymer, the amountof polymer relative to the total weight of the spray dry dispersionranges from about 0.1% to 99% by weight. Unless otherwise specified,percentages of drug, polymer and other excipients as described within adispersion are given in weight percentages. The amount of polymer istypically at least about 20%, and preferably at least about 30%, forexample, at least about 35%, at least about 40%, at least about 45%, orabout 50% (e.g., 49.5%). The amount is typically about 99% or less, andpreferably about 80% or less, for example about 75% or less, about 70%or less, about 65% or less, about 60% or less, or about 55% or less. Inone embodiment, the polymer is in an amount of up to about 50% of thetotal weight of the dispersion (and even more specifically, betweenabout 40% and 50%, such as about 49%, about 49.5%, or about 50%). HPMCand HPMCAS are available in a variety of grades from ShinEtsu, forexample, HPMCAS is available in a number of varieties, including AS-LF,AS-MF, AS-HF, AS-LG, AS-MG, AS-HG. Each of these grades vary with thepercent substitution of acetate and succinate.

In some embodiments, substantially amorphous Compound 2 and polymer arepresent in roughly equal amounts, for example each of the polymer andthe drug make up about half of the percentage weight of the dispersion.For example, the polymer is present in about 49.5% and the drug ispresent in about 50%.

In some embodiments, substantially amorphous Compound 2 and the polymercombined represent 1% to 20% w/w total solid content of the non-spraydry dispersion prior to spray drying. In some embodiments, substantiallyamorphous Compound 2 and the polymer combined represent 5% to 15% w/wtotal solid content of the non-spray dry dispersion prior to spraydrying. In some embodiments, substantially amorphous Compound 2 and thepolymer combined represent about 11% w/w total solid content of thenon-spray dry dispersion prior to spray drying.

In some embodiments, the dispersion further includes other minoringredients, such as a surfactant (e.g., SLS). In some embodiments, thesurfactant is present in less than about 10% of the dispersion, forexample less than about 9%, less than about 8%, less than about 7%, lessthan about 6%, less than about 5%, less than about 4%, less than about3%, less than about 2%, about 1%, or about 0.5%.

In embodiments including a polymer, the polymer should be present in anamount effective for stabilizing the spray dry dispersion. Stabilizingincludes inhibiting or preventing, the crystallization of substantiallyamorphous Compound 2. Such stabilizing would inhibit the conversionCompound 2 from amorphous to crystalline form. For example, the polymerwould prevent at least a portion (e.g., about 5%, about 10%, about 15%,about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about50%, about 55%, about 60%, about 65%, about 70%, about 75%, or greater)of Compound 2 from converting from an amorphous to a crystalline form.Stabilization can be measured, for example, by measuring the glasstransition temperature of the spray dry dispersion, measuring the rateof relaxation of the amorphous material, or by measuring the solubilityor bioavailability of Compound 2.

Suitable polymers for use in combination with Compound 2, for example toform a spray dry dispersion such as an amorphous spray dry dispersion,should have one or more of the following properties:

The glass transition temperature of the polymer should have atemperature of no less than about 10-15° C. lower than the glasstransition temperature of substantially amorphous Compound 2.Preferably; the glass transition temperature of the polymer is greaterthan the glass transition temperature of substantially amorphousCompound 2, and in general at least 50° C. higher than the desiredstorage temperature of the drug product. For example, at least about100° C., at least about 105° C., at least about 105° C., at least about110° C., at least about 120° C., at least about 130° C., at least about140° C., at least about 150° C., at least about 160° C., at least about160° C., or greater.

The polymer should be relatively non-hygroscopic. For example, thepolymer should, when stored under standard conditions, absorb less thanabout 10% water, for example, less than about 9%, less than about 8%,less than about 7%, less than about 6%, or less than about 5%, less thanabout 4%, or less than about 3% water. Preferably the polymer will, whenstored under standard conditions, be substantially free of absorbedwater.

The polymer should have similar or better solubility in solventssuitable for spray drying processes relative to that of Compound 2. Inpreferred embodiments, the polymer will dissolve in one or more of thesame solvents or solvent systems as Compound 2. It is preferred that thepolymer is soluble in at least one non-hydroxy containing solvent suchas methylene chloride, acetone, or a combination thereof.

The polymer, when combined with substantially amorphous Compound 2, forexample in a spray dry dispersion or in a liquid suspension, shouldincrease the solubility of Compound 2 in aqueous and physiologicallyrelative media either relative to the solubility of Compound 2 in theabsence of polymer or relative to the solubility of Compound 2 whencombined with a reference polymer. For example, the polymer couldincrease the solubility of amorphous Compound 2 by reducing the amountof amorphous Compound 2 that converts to crystalline Compound 2, eitherfrom a solid amorphous dispersion or from a liquid suspension.

The polymer should decrease the relaxation rate of the amorphoussubstance.

The polymer should increase the physical and/or chemical stability ofsubstantially amorphous Compound 2.

The polymer should improve the manufacturability of substantiallyamorphous Compound 2.

The polymer should improve one or more of the handling, administrationor storage properties of substantially amorphous Compound 2.

The polymer should not interact unfavorably with other pharmaceuticalcomponents, for example excipients.

The suitability of a candidate polymer (or other component) can betested using the spray drying methods (or other methods) describedherein to form an amorphous composition. The candidate composition canbe compared in terms of stability, resistance to the formation ofcrystals, or other properties, and compared to a reference preparation,e.g., a preparation of neat amorphous Compound 2 or crystalline Compound2. For example, a candidate composition could be tested to determinewhether it inhibits the time to onset of solvent mediatedcrystallization, or the percent conversion at a given time undercontrolled conditions, by at least 50%, 75%, 100%, or 110% as well asthe reference preparation, or a candidate composition could be tested todetermine if it has improved bioavailability or solubility relative tocrystalline Compound 2.

Surfactants

The spray dry dispersion may include a surfactant. A surfactant orsurfactant mixture would generally decrease the interfacial tensionbetween the spray dry dispersion and an aqueous medium. An appropriatesurfactant or surfactant mixture may also enhance aqueous solubility andbioavailability of Compound 2 from a spray dry dispersion. Thesurfactants for use in connection with the present invention include,but are not limited to, sorbitan fatty acid esters (e.g., Spans®),polyoxyethylene sorbitan fatty acid esters (e.g., Tweens®), sodiumlauryl sulfate (SLS), sodium dodecylbenzene sulfonate (SDBS) dioctylsodium sulfosuccinate (Docusate), dioxycholic acid sodium salt (DOSS),Sorbitan Monostearate, Sorbitan Tristearate, hexadecyltrimethyl ammoniumbromide (HTAB), Sodium N-lauroylsarcosine, Sodium Oleate, SodiumMyristate, Sodium Stearate, Sodium Palmitate, Gelucire 44/14,ethylenediamine tetraacetic acid (EDTA), Vitamin E d-alpha tocopherylpolyethylene glycol 1000 succinate (TPGS), Lecithin, MW 677-692,Glutanic acid monosodium monohydrate, Labrasol, PEG 8 caprylic/capricglycerides, Transcutol, diethylene glycol monoethyl ether, SolutolHS-15, polyethylene glycol/hydroxystearate, Taurocholic Acid, PluronicF68, Pluronic F108, and Pluronic F127 (or any otherpolyoxyethylene-polyoxypropylene co-polymers (Pluronics®) or saturatedpolyglycolized glycerides (Gelucirs®)). Specific example of suchsurfactants that may be used in connection with this invention include,but are not limited to, Span 65, Span 25, Tween 20, Capryol 90, PluronicF108, sodium lauryl sulfate (SLS), Vitamin E TPGS, pluronics andcopolymers. SLS is generally preferred.

The amount of the surfactant (e.g., SLS) relative to the total weight ofthe spray dry dispersion may be between 0.1-15%. Preferably, it is fromabout 0.5% to about 10%, more preferably from about 0.5 to about 5%,e.g., about 0.5 to 4%, about 0.5 to 3%, about 0.5 to 2%, about 0.5 to1%, or about 0.5%.

In certain embodiments, the amount of the surfactant relative to thetotal weight of the spray dry dispersion is at least about 0.1%,preferably about 0.5%. In these embodiments, the surfactant would bepresent in an amount of no more than about 15%, and preferably no morethan about 12%, about 11%, about 10%, about 9%, about 8%, about 7%,about 6%, about 5%, about 4%, about 3%, about 2% or about 1%. Anembodiment wherein the surfactant is in an amount of about 0.5% byweight is preferred.

Candidate surfactants (or other components) can be tested forsuitability for use in the invention in a manner similar to thatdescribed for testing polymers.

Methods for Making the Pharmaceutical Compositions

The pharmaceutical compositions of the invention can be produced by, wetgranulation, compacting or compressing an admixture or composition, forexample, a powder or granules, under pressure to form a stablethree-dimensional shape (e.g., a tablet). As used herein, “tablet”includes compressed pharmaceutical dosage unit forms of all shapes andsizes, whether coated or uncoated.

The term “tablet” as used herein refers to a physically discrete unit ofagent appropriate for the patient to be treated. In general, a compactedmixture has a density greater than that of the mixture prior tocompaction. A dosage tablet of the invention can have almost any shapeincluding concave and/or convex faces, rounded or angled corners, and arounded to rectilinear shape. In some embodiments, the compressedtablets of the invention comprise a rounded tablet having flat faces.The tablets of the invention can be prepared by any compaction andcompression method known by persons of ordinary skill in the art offorming compressed solid pharmaceutical dosage forms. In particularembodiments, the formulations provided herein may be prepared usingconventional methods known to those skilled in the field ofpharmaceutical formulation, as described, e.g., in pertinent textbooks.See, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed.,Lippincott Williams & Wilkins, Baltimore, Md. (2003); Ansel et al.,Pharmaceutical Dosage Forms And Drug Delivery Systems, 7th Edition,Lippincott Williams & Wilkins, (1999); The Handbook of PharmaceuticalExcipients, 4^(th) edition, Rowe et al., Eds., American PharmaceuticalsAssociation (2003); Gibson, Pharmaceutical Preformulation AndFormulation, CRC Press (2001), these references hereby incorporatedherein by reference in their entirety.

Granulation and Compression

In some embodiments, the ingredients are weighed according to theformula set herein. Next, all of the intragranular ingredients aresifted and mixed well. The ingredients can be lubricated with a suitablelubricant, for example, magnesium stearate. The next step can comprisecompaction/slugging of the powder admixture and sized ingredients. Next,the compacted or slugged blends are milled into granules and sifted toobtain the desired size. Next, the granules can be further lubricatedwith, for example, magnesium stearate. Next the granular composition ofthe invention can be compressed on suitable punches into variouspharmaceutical formulations in accordance with the invention. Optionallythe tablets can be coated with a film, colorant or other coating.

Another aspect of the invention provides a method for producing apharmaceutical composition comprising providing an admixture of acomposition comprising Compound 1 Form I, a solid dispersion comprisingsubstantially amorphous Compound 2 and one or more excipients selectedfrom: a filler, a diluent, a binder, a surfactant, a lubricant, adisintegrant, and compressing the composition into a tablet having adissolution of at least about 50% in about 30 minutes.

In another embodiment, a wet granulation process is performed to yieldthe pharmaceutical formulation of the invention from an admixture ofpowdered and liquid ingredients. For example, a pharmaceuticalcomposition comprising an admixture of a composition comprising Compound1 Form I, a solid dispersion comprising substantially amorphous Compound2, and one or more excipients selected from: a filler, a binder, asurfactant, or a disintegrant, are weighed as per the formula setherein. Next, all of the intragranular ingredients are sifted and mixedin a high shear or low shear granulator using water or water with asurfactant or water with a binder or water with a surfactant and abinder to granulate the powder blend. A fluid other than water can alsobe used with or without surfactant and/or binder to granulate the powderblend. Next, the wet granules can optionally be milled using a suitablemill. Next, water may optionally be removed from the admixture by dryingthe ingredients in any suitable manner. Next, the dried granules canoptionally be milled to the required size. Next, extra granularexcipients can be added by blending (for example a filler, a diluent,and a disintegrant). Next, the sized granules can be further lubricatedwith magnesium stearate and a disintegrant, for example, croscarmellosesodium. Next the granular composition of the invention can be sifted forsufficient time to obtain the correct size and then compressed onsuitable punches into various pharmaceutical formulations in accordancewith the invention. Optionally, the tablets can be coated with a film,colorant or other coating. Surprisingly, wet granulation can be carriedout without substantial loss of the solid state forms of Compound 1 FormI or substantially amorphous Compound 2.

In a particularly favored embodiment, the pharmaceutical compositions ofthe present invention are prepared by a continuous twin screw wetgranulation (TSWG) process. Continuous manufacturing delivers highquality and highly consistent product with on-line monitoring andcontrol. Continuous manufacturing also facilitates quality by designdevelopment with a “data rich” design space and an easier to understandimpact of upstream variables on the downstream process and final productquality. In addition, the pharmaceutical compositions of the presentinvention can be finalized early on commercial scale equipment whichavoids scale-up risks and formulation changes late in development.Finally, continuous manufacturing has commercial manufacturingadvantages such as improved process control, reduced product handling,and real time release efficiencies. The overall result is a more robust,controllable, and scalable process that has fewer process checksresulting in increased product quality and therefore greater patientsafety. These advantages address Janet Woodcock's (director of theCenter for Drug Evaluation and Research (CDER)) concerns that chemistry,manufacturing, and controls (CMC) won't be able to keep up with rapidclinical development of highly effective therapies (“What we are seeingis that often the rate limiting step is going to be manufacturing,” Jul.24, 2013 Friends of Cancer hosted congressional briefing “Answering aCompelling Need: Expediting Life-Saving Treatments to Patients” todiscuss the Food and Drug Administration's Breakthrough TherapyDesignation).

For example, high shear granulation (HSG), a common granulationtechnique is well known for the risk of over-granulation and poorprocess control. Scale-up of this process is very challenging andinvolves significant risk. Changing from a HSG process to a continuousTSWG process, allows scale-up using the same equipment to producedifferent batch sizes, by running for a longer time. This eliminates thescale-up risk commonly encountered with other granulation processes.Additionally, it was found that the TSWG process is more robust, beingless sensitive to over-granulation. As can be seen in FIG. 3 for aCompound 1 tablet, the HSG process showed significant dissolutionslow-down with increasing water content, while the TSWG process did notshow a change for a similar range of water addition. Surprisingly, noperformance changes were found with the tablet formulations comprisingCompound 1 between 45-55 percent by weight and the tablet formulationscomprising Compound 1 between 60-70 percent by weight using the twinscrew wet granulation process. This was not the case with the HSGprocess. Additionally, this continuous and increased product qualityprocess addresses a common complaint by the FDA regarding the lack ofdrug availability for patients in need thereof.

In one embodiment the continuous process starts with feeding individualexcipients, Compound 1, and Compound 2 into a continuous in-line blenderthrough loss-in-weight feeding. From this blender, the material iscontinuously conveyed and processed through twin screw wet granulation,drying, milling, extra-granular excipient addition, blending,compression and film coating.

For example, in one embodiment, a tablet comprising Compound 1 andCompound 2 may be prepared continuously according to the flow chartdepicted in FIG. 26.

Each of the ingredients of this exemplary admixture is described aboveand in the Examples below. Furthermore, the admixture can compriseoptional additives, such as, one or more colorants, one or more flavors,and/or one or more fragrances as described above and in the Examplesbelow. In some embodiments, the relative concentrations (e.g., wt %) ofeach of these ingredients (and any optional additives) in the admixtureare also presented above and in the Examples below. The ingredientsconstituting the admixture can be provided sequentially or in anycombination of additions; and, the ingredients or combination ofingredients can be provided in any order. In one embodiment, thelubricant is the last component added to the admixture.

In another embodiment, the admixture comprises a composition of Compound1 Form I, a solid dispersion of substantially amorphous Compound 2, andany one or more of the excipients; a binder, a surfactant, a diluent, alubricant, a disintegrant, and a filler, wherein each of theseingredients is provided in a powder form (e.g., provided as particleshaving a mean or average diameter, measured by light scattering, of 250μm or less (e.g., 150 μm or less, 100 μm or less, 50 μm or less, 45 μmor less, 40 μm or less, or 35 μm or less)).

In another embodiment, compressing the admixture into a tablet isaccomplished by filling a form (e.g., a mold) with the admixture andapplying pressure to admixture. This can be accomplished using a diepress or other similar apparatus. In some embodiments, the admixture ofCompound 1 Form I, a solid dispersion comprising substantially amorphousCompound 2, and excipients can be first processed into granular form.The granules can then be sized and compressed into tablets or formulatedfor encapsulation according to known methods in the pharmaceutical art.It is also noted that the application of pressure to the admixture inthe form can be repeated using the same pressure during each compressionor using different pressures during the compressions. In anotherexample, the admixture of powdered ingredients or granules can becompressed using a die press that applies sufficient pressure to form atablet having a dissolution of about 50% or more at about 30 minutes(e.g., about 55% or more at about 30 minutes or about 60% or more atabout 30 minutes). For instance, the admixture is compressed using a diepress to produce a tablet hardness of at least about 5 kP (at leastabout 5.5 kP, at least about 6 kP, at least about 7 kP, at least about10 kP, or at least 15 kP). In some instances, the admixture iscompressed to produce a tablet hardness of between about 5 and 20 kP.

In some embodiments, tablets comprising a pharmaceutical composition asdescribed herein can be coated with about 3.0 wt % of a film coatingcomprising a colorant by weight of the tablet. In certain instances, thecolorant suspension or solution used to coat the tablets comprises about20% w/w of solids by weight of the colorant suspension or solution. Instill further instances, the coated tablets can be labeled with a logo,other image or text.

In another embodiment, the method for producing a pharmaceuticalcomposition comprises providing an admixture of a solid forms, e.g. anadmixture of powdered and/or liquid ingredients, the admixturecomprising Compound 1 Form I, a solid dispersion comprisingsubstantially amorphous Compound 2, and one or more excipients selectedfrom: a binder, a diluent, a surfactant, a lubricant, a disintegrant,and a filler; mixing the admixture until the admixture is substantiallyhomogenous, and compressing or compacting the admixture into a granularform. Then the granular composition comprising Compound 1 Form I and asolid dispersion comprising substantially amorphous Compound 2 can becompressed into tablets or formulated into capsules as described aboveor in the Examples below. Alternatively, methods for producing apharmaceutical composition comprises providing an admixture of Compound1 Form I, a solid dispersion comprising substantially amorphous Compound2, and one or more excipients, e.g. a binder, a diluent, a surfactant, alubricant, a disintegrant, and a filler; mixing the admixture until theadmixture is substantially homogenous, and compressing/compacting theadmixture into a granular form using a high shear wet granule compactionprocess as set forth in the Examples below. Pharmaceutical formulations,for example a tablet as described herein, can be made using the granulesprepared incorporating Compound 1 Form I and a solid dispersioncomprising substantially amorphous Compound 2 in addition to theselected excipients described herein.

In some embodiments, the admixture is mixed by stirring, blending,shaking, or the like using hand mixing, a mixer, a blender, anycombination thereof, or the like. When ingredients or combinations ofingredients are added sequentially, mixing can occur between successiveadditions, continuously throughout the ingredient addition, after theaddition of all of the ingredients or combinations of ingredients, orany combination thereof. The admixture is mixed until it has asubstantially homogenous composition.

In another embodiment, the present invention comprises jet milling apharmaceutical composition comprising Compound 1 Form I and a soliddispersion comprising substantially amorphous Compound 2 in a suitable,conventional milling apparatus using air pressure suitable to produceparticles having a significant particle size fraction between 0.1microns and 50 microns. In another embodiment, the particle size isbetween 0.1 microns and 20 microns. In another embodiment, the particlessize is between 0.1 microns and 10 microns. In another embodiment, theparticle size is between 1.0 microns and 5 microns. In still anotherembodiment, the pharmaceutical composition has a particle size D50 of2.0 microns.

The formulations of the present invention provide a fixed dosage of twoAPIs for the effective treatment of cystic fibrosis, a combination thathas received one of only two Breakthrough Therapy Designation from theFDA, and does so with surprising stability as measured by the small lossof the amorphous solid form of Compound 2. FIG. 4 depicts the smallamount of crystallinity of Compound 2 over time in PC-XVII at 50° C.after pre-equilibration at 60% relative humidity. Even after close to1000 hours under these conditions, less than 5% by weight of Compound 2has crystallized. FIG. 5 shows for PC-XVII that even at the highertemperature of 60° C. after pre-equilibrating at 60% relative humidity,at close to 1000 hours under these conditions, less than 10% by weightof Compound 2 has crystallized. FIGS. 6 and 7 show similar results forPC-XIX. The present formulations, therefore, provide the convenience ofa fixed dosage of two breakthrough API's in a surprisingly stablepharmaceutical composition. Such formulations increase patientcompliance which directly relates to the effective treatment ofdiseases.

Dosage forms prepared as above can be subjected to in vitro dissolutionevaluations according to Test 711 “Dissolution” in United StatesPharmacopoeia 29, United States Pharmacopeial Convention, Inc.,Rockville, Md., 2005 (“USP”), to determine the rate at which the activesubstance is released from the dosage forms. The content of activesubstance and the impurity levels are conveniently measured bytechniques such as high performance liquid chromatography (HPLC).

In some embodiments, the invention includes use of packaging materialssuch as containers and closures of high-density polyethylene (HDPE),low-density polyethylene (LDPE) and or polypropylene and/or glass,glassine foil, aluminum pouches, and blisters or strips composed ofaluminum or high-density polyvinyl chloride (PVC), optionally includinga desiccant, polyethylene (PE), polyvinylidene dichloride (PVDC),PVC/PE/PVDC, and the like. These package materials can be used to storethe various pharmaceutical compositions and formulations in a sterilefashion after appropriate sterilization of the package and its contentsusing chemical or physical sterilization techniques commonly employed inthe pharmaceutical arts.

Methods for Administering the Pharmaceutical Compositions

In one aspect, the pharmaceutical compositions of the invention can beadministered to a patient once daily or about every twenty four hours.Alternatively, the pharmaceutical compositions of the invention can beadministered to a patient twice daily. Alternatively, the pharmaceuticalcomposition of the invention can be administered about every twelvehours. These pharmaceutical compositions are administered as oralformulations containing about 25 mg, 50 mg, 100 mg, 125 mg, 150 mg, 200mg, 250 mg, 300 mg, or 400 mg of Compound 1 Form I; and about 25 mg, 50mg, 100 mg, 125 mg, 150 mg, 200 mg, or 250 mg of substantially amorphousCompound 2. In this aspect, in addition to Compound 1 Form I andsubstantially amorphous Compound 2, the pharmaceutical compositionscomprise a filler; a disintegrant; a surfactant; a binder; and alubricant (depending on whether the pharmaceutical composition is agranule or a tablet). For instance, a dose of 400 mg of Compound 1 FormI, may comprise two tablets of the invention each containing 200 mg ofCompound 1 Form I. A dose of 250 mg of substantially amorphous Compound2, may comprise two tablets of the invention each containing 125 mg ofsubstantially amorphous Compound 2.

It will also be appreciated that the compound and pharmaceuticallyacceptable compositions and formulations of the invention can beemployed in combination therapies; that is, Compound 1 Form I and asolid dispersion of substantially amorphous Compound 2 andpharmaceutically acceptable compositions thereof can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures.

In one embodiment, the additional therapeutic agent is selected from amucolytic agent, bronchodialator, an antibiotic, an anti-infectiveagent, an anti-inflammatory agent, a compound that induces CFTR activityother than Compound 1 Form I and substantially amorphous Compound 2, ora nutritional agent.

In one embodiment, the additional agent is(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.In another embodiment, the additional agent is4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid. In another embodiment, the additionalagent is selected from Table 1:

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

In another embodiment, the additional agent is any combination of theabove agents. For example, the combination may comprise a pharmaceuticalcomposition or tablet of the present invention comprising Compound 1Form I and a solid dispersion of substantially amorphous Compound 2, andthe additional therapeutic agent is(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide.In another example, the combination may comprise a pharmaceuticalcomposition or tablet of the present invention comprising Compound 1Form I and a solid dispersion of substantially amorphous Compound 2, andthe additional therapeutic agent is4-(3-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)isoquinolin-1-yl)benzoic acid. In another example, the combination maycomprise a pharmaceutical composition or tablet of the present inventioncomprising Compound 1 Form I and a solid dispersion of substantiallyamorphous Compound 2, and the additional therapeutic agent is any one ofthe compounds from Table 1, i.e. compounds 1 through 14 of Table 1, orany combination thereof.

In another embodiment, the additional agent is selected from Table 1:

TABLE 1 Compounds disclosed in U.S. Pat. No. 7,407,976 (Col 13, ln35-col 66, ln 67; Compounds 1-100 in Table 1 at col 67, ln 1-col 127, ln42) Compounds disclosed in U.S. Pat. No. 7,645,789 (Col 16, ln 52-col50, ln 22; Compounds 1-322 in Table 1 at col 50, ln 24-col 167, ln 42)Compounds disclosedin U.S. Pat. No. 7,659,268 (Col 16, ln 20-col 70, ln52; Compounds 1-528 in Table 1 at col 70, ln 53-col 331, ln 34)Compounds disclosed in U.S. Pat. No. 7,671,221 (Col 16, ln 12-col 54, ln48; Compounds 1-1216 in Table 1 at col 54, ln 49-col 699, ln 27)Compounds disclosed in U.S. Pat. No. 7,691,902 (Col 16, ln 11-col 54, ln29; Compounds 1-959 in Table 1 at col 54, ln 29-col 683, ln 44)Compounds disclosed in U.S. Pat. No. 7,741,321 (Col 16, ln 25-col 72, ln17; Compounds 1-422 in Table 1 at col 72, ln 20-col 279, ln 15)Compounds disclosed in U.S. Pat. No. 7,754,739 (Col 16, ln 1-col 22, ln47; Compounds 1-2 in Table 1 at col 18, ln 26-65) Compounds disclosed inU.S. Pat. No. 7,776,905 (Col 16, ln 23-col 38, ln 40; Compounds 1-306 inTable 1 at col 38, ln 45-col 96, ln 40) Compounds disclosed in U.S. Pat.No. 7,973,169 (Col 9, ln 16-col 40, ln 40; Compounds 1-289 in Table 1 atcol. 40, ln 41-col 289, ln 39) Compounds disclosed in U.S. Pat. No.7,977,322 (Col 6, ln 26-col 37, ln 47; Compounds 1-498 in Table 1 at col37, ln 50-col 141, ln 40) Compounds disclosed in U.S. Pat. No. 7,999,113(Col 6, ln 13-col 10, ln 67; Compounds 1-13 in Table 1 at col 11, ln5-col 13, ln 65) Compounds disclosed in U.S. Pat. No. 8,227,615 (Col 6,ln 10-col 29, ln 66; Compounds 1-78 in Table 1 at col 30, ln 1-col 46,ln 48) Compounds disclosed in U.S. Pat. No. 8,299,099 (Col 6, ln 10-col34, ln 18; Compounds 1-47 in Table 1 at col 34, ln 20-col 42, ln 35)Compounds disclosed in US Published Application No. 2006-0052358(Paragraphs [0034]- [0056]; [0077]-[0240]; Compounds 1-320 in Table 1 atparagraph [0241]) Compounds disclosed in US Published Application No.2009-0143381 (Paragraphs [0102]- [0263]; Compounds 1-28 in Table 1 atparagraph [0264]) Compounds disclosed in US Published Application No.2009-0170905 (Paragraphs [0012]- [0013]; [0030]-[0051]) Compoundsdisclosed in US Published Application No. 2009-0253736 (Paragraphs[0031]- [0162]; Compounds 1-15 in Table 1 at paragraph [0163]) Compoundsdisclosed in US Published Application No. 2011-0263654 (Paragraphs[0012]- [0013]; [0066]-[0141]) Compounds disclosed in US PublishedApplication No. 2011-0251253 (Paragraphs [0012]- [0013]; [0054]-[0079])Compounds disclosed in PCT application WO2008141119 (Paragraphs[0100]-[0339]; Compounds 1-117 in Table 1 at paragraph [0340]) Compoundsdisclosed in U.S. application No. 11/047,361 Compounds disclosed in USPublished Application No. 2013-0116238 (Paragraphs [0028]- [0044];[0117]-[0128]), or combinations thereof.

In another embodiment, the additional agent is selected from Table 2:

TABLE 2 Compounds disclosed in US Published Application No. 2005-0113423(Paragraph [00146]; Compounds IA-1-IA-136 and Compounds I-1-I-21 inTables 1 and 2 at paragraphs [0391]-[0392]) Compounds disclosed in USPublished Application No. 2005-0059687 (Paragraphs [00100]-[00101];Compounds 1-405 in Table 1 at paragraph [0169]) Compounds 1-108disclosed in U.S. Pat. No. 7,598,412 (Col 22, ln 14-col 79, ln 20;Table 1) Compounds 1-485 disclosed in U.S. Pat. No. 7,495,103 (Col 51,ln 1-col 63, ln 43; Table 1) Compounds 1-718 disclosed in U.S. Pat. No.8,354,427 (Col 51, ln 3-col 71, ln 46; Table 1) Compounds 1-233disclosed in US Published Application No. 2007-0105833 (Paragraph[00145]; Table 1) Compounds 1-26 disclosed in U.S. Pat. No. 8,242,149(Col 46, ln 47-col 57, ln 37; Table 1) Compounds 1-18 disclosed in U.S.Pat. No. 8,314,256 (Col 21, ln 1-col 26, ln 19) Compounds 1-14 disclosedin U.S. Pat. No. 8,399,479 (Col 36, ln 20-col 38, ln 40; Table 1)Compounds 1-18 disclosed in U.S. Pat. No. 8,188,283 (Col 38, ln 43-col43, ln 36; Table 1) Compounds 1-16 disclosed in US Published ApplicationNo. 2010-0249180 (Paragraph [0173]; Table 1) Compounds 1-19 disclosed inUS Published Application No. 2011-0008259 (Paragraph [0172]; Table 1)Compounds 1-129 disclosed in U.S. Pat. No. 8,367,660 (Col 57, ln 31-col81, ln 24; Table 1)

In one embodiment, the additional therapeutic agent is an antibiotic.Exemplary antibiotics useful herein include tobramycin, includingtobramycin inhaled powder (TIP), azithromycin, cayston, aztreonam,including the aerosolized form of aztreonam, amikacin, includingliposomal formulations thereof, ciprofloxacin, including formulationsthereof suitable for administration by inhalation, levoflaxacin,including aerosolized formulations thereof, and combinations of twoantibiotics, e.g., fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen phosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a compound that augmentsor induces CFTR activity other than Compound 1 Form I or a soliddispersion comprising substantially amorphous Compound 2, i.e., an agentthat has the effect of inducing or augmenting CFTR activity. Exemplarysuch agents include ataluren (“PTC 124®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), and cobiprostone (7-{(2R, 4aR, 5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid).

In another embodiment, the additional agent is a nutritional agent.Exemplary nutritional agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPaugmenters or inducers such as rolipram, sildenafil, milrinone,tadalafil, amrinone, isoproterenol, albuterol, and almeterol,deoxyspergualin, HSP 90 inhibitors, HSP 70 inhibitors, proteosomeinhibitors such as epoxomicin, lactacystin, etc.

In another embodiment, the additional agent is a compound selected from3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;5-amino-6′-methyl-3-trifluoromethyl-[2,3]bipyridinyl-6-carboxylic acid(3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-cyclopropyl-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide;3-amino-6-methoxy-N-(3,3,3-trifluoro-2-hydroxy-2-(trifluoromethyl)propyl)-5-(trifluoromethyl)picolinamide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((S-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-aide;3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylicacid ((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(2,4-dichloro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylicacid ((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;(S)-3-amino-6-ethoxy-N-(3,3,3-trifluoro-2-hydroxy-2-methylpropyl)-5-(trifluoromethyl)picolinamide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-methoxy-5-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-6-(4-fluoro-phenyl)-5-trifluoromethyl-pyridine-2-carboxylic acid(3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((S)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide;3-amino-5,6-bis-trifluoromethyl-pyridine-2-carboxylic acid((R)-3,3,3-trifluoro-2-hydroxy-2-methyl-propyl)-amide, orpharmaceutically acceptable salts thereof. In another embodiment, theadditional agent is a compound disclosed in U.S. Pat. No. 8,247,436 andInternational PCT Publication WO 2011113894, incorporated herein intheir entirety by reference.

In another embodiment, the additional agent may be an epithelial sodiumchannel (ENac) modulator disclosed in PCT publications WO2012035158,WO2009074575, WO2011028740, WO2009150137, WO2011079087, or WO2008135557,incorporated herein in their entirety by reference.

In other embodiments, the additional agent is a compound disclosed in WO2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740, incorporated herein in their entirety by reference. Inanother embodiment, the additional agent is a benzo[c]quinoliziniumderivative that exhibits CFTR inducing or augmenting activity or abenzopyran derivative that exhibits CFTR inducing or augmentingactivity. In another embodiment, the additional agent is a compounddisclosed in U.S. Pat. Nos. 7,202,262, 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO02006044503, WO2006044502, orWO2004091502, incorporated herein in their entirety by reference. Inanother embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256,WO2006127588, or WO2007044560, incorporated herein in their entirety byreference.

In one embodiment, 400 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 400 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be achieved by administering twotablets each containing 200 mg of Compound 1 Form I, and 125 mg ofsubstantially amorphous Compound 2. The duration of administration maycontinue until amelioration of the disease is achieved or until asubject's physician advises, e.g. duration of administration may be lessthan a week, 1 week, 2 weeks, 3 weeks, four weeks (28 days), or a monthor longer. In one embodiment, two tablets each comprising 200 mg ofCompound 1 Form I, and 125 mg of substantially amorphous Compound 2 maybe administered to the patient per day. In a further embodiment, the twotablets may be administered at the same time or at different timesduring the day. In a further embodiment, one tablet is administeredevery 12 hours.

In one embodiment, 400 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of two tablets each containing 200 mg of Compound 1Form I, and 250 mg of substantially amorphous Compound 2. In oneembodiment a tablet is administered once every 12 hours. In anotherembodiment, the dosage amount may also be achieved by administering twotablets, each containing 100 mg of Compound 1 Form I and 125 mg ofsubstantially amorphous Compound 2, every 12 hours. In anotherembodiment, the dosage amounts may also be achieved by administeringCompound 1 Form I and substantially amorphous Compound 2 in separatetablets. For example, the dosage amounts may be achieved byadministering two tablets containing 200 mg of Compound 1 Form I, andfour tablets containing 125 mg of substantially amorphous Compound 2 ortwo tablets containing 150 mg of substantially amorphous Compound 2 andtwo tablets containing 100 mg of substantially amorphous Compound 2. Theduration of administration may continue until amelioration of thedisease is achieved or until a subject's physician advises, e.g.duration of administration may be less than a week, 1 week, 2 weeks, 3weeks, four weeks (28 days), or a month or longer. In one embodiment,two tablets comprising 200 mg of Compound 1 Form I, and four tabletscomprising 125 mg of substantially amorphous Compound 2 may beadministered to the patient per day. In one embodiment, two tabletscomprising 200 mg of Compound 1 Form I may be administered to thepatient per day, and two tablets comprising 150 mg and 100 mg ofsubstantially amorphous Compound 2 may be administered to the patienttwice per day. In a further embodiment, the two tablets may beadministered at the same time or at different times during the day. In afurther embodiment, one tablet comprising 200 mg of Compound 1 isadministered every 12 hours, and two tablets comprising 150 mg and 100mg of substantially amorphous Compound 2 are administered every 12hours.

In one embodiment, 300 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 300 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be achieved by administering twotablets each containing 150 mg of Compound 1 Form I, and 125 mg ofsubstantially amorphous Compound 2. The duration of administration maycontinue until amelioration of the disease is achieved or until asubject's physician advises, e.g. duration of administration may be lessthan a week, 1 week, 2 weeks, 3 weeks, four weeks (28 days), or a monthor longer. In one embodiment, two tablets each comprising 150 mg ofCompound 1 Form I, and 125 mg of substantially amorphous Compound 2 maybe administered to the patient per day. In a further embodiment, the twotablets may be administered at the same time or at different timesduring the day. In a further embodiment, one tablet is administeredevery 12 hours.

In one embodiment, 600 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 600 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be achieved by administering twotablets, each containing 150 mg of Compound 1 Form I, and 125 mg ofsubstantially amorphous Compound 2, every 12 hours. The duration ofadministration may continue until amelioration of the disease isachieved or until a subject's physician advises, e.g. duration ofadministration may be less than a week, 1 week, 2 weeks, 3 weeks, fourweeks (28 days), or a month or longer. In one embodiment, four tabletseach comprising 150 mg of Compound 1 Form I, and 125 mg of substantiallyamorphous Compound 2 may be administered to the patient per day. In afurther embodiment, the four tablets may be administered at the sametime or at different times during the day. In a further embodiment, twotablet is administered every 12 hours.

In one embodiment, 800 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 800 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be achieved by administering fourtablets each containing 200 mg of Compound 1 Form I, and 125 mg ofsubstantially amorphous Compound 2. The duration of administration maycontinue until amelioration of the disease is achieved or until asubject's physician advises, e.g. duration of administration may be lessthan a week, 1 week, 2 weeks, 3 weeks, four weeks (28 days), or a monthor longer. In one embodiment, four tablets each comprising 200 mg ofCompound 1 Form I, and 125 mg of substantially amorphous Compound 2 maybe administered to the patient per day. In a further embodiment, thefour tablets may be administered at the same time or at different timesduring the day. In a further embodiment, two tablets are administeredper dosing occasion, and there are two dosing occasions per day. In afurther embodiment, 800 mg of Compound 1 and 500 mg of Compound 2 areadministered to the patient by administering two tablets each comprising200 mg of Compound 1 and 125 mg of Compound 2 twice a day (BID). In afurther embodiment, 800 mg of Compound 1 and 500 mg of Compound 2 areadministered to the patient by administering two tablets each comprising200 mg of Compound 1 and 125 mg of Compound 2 every 12 hours (q12h).

In one embodiment, 600 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 600 mg of Compound 1 Form I and 250 mg ofsubstantially amorphous Compound 2 may be achieved by administeringthree tablets each containing 200 mg of Compound 1 Form I, and 83.3 mgof substantially amorphous Compound 2. The duration of administrationmay continue until amelioration of the disease is achieved or until asubject's physician advises, e.g. duration of administration may be lessthan a week, 1 week, 2 weeks, 3 weeks, four weeks (28 days), or a monthor longer. In one embodiment, three tablets each comprising 200 mg ofCompound 1 Form I, and 83.3 mg of substantially amorphous Compound 2 maybe administered to the patient per day. In a further embodiment, thethree tablets may be administered at the same time or at different timesduring the day. In a further embodiment, three tablets are administeredat the same time.

In one embodiment, 600 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be administered to a subject inneed thereof. In these embodiments, the dosage amounts may be achievedby administration of one or more tablets of the invention. For example,administration of 600 mg of Compound 1 Form I and 500 mg ofsubstantially amorphous Compound 2 may be achieved by administeringthree tablets each containing 200 mg of Compound 1 Form I, and 83.3 mgof substantially amorphous Compound 2, followed by two additionaltablets each comprising 125 mg of Compound 2. The duration ofadministration may continue until amelioration of the disease isachieved or until a subject's physician advises, e.g. duration ofadministration may be less than a week, 1 week, 2 weeks, 3 weeks, fourweeks (28 days), or a month or longer. In one embodiment, 600 mg ofCompound 1 may be administered daily (qd) and 250 mg of Compound 2administered twice a day (bid) by administering three tablets eachcomprising 200 mg of Compound 1 Form I, and 83.3 mg of substantiallyamorphous Compound 2 daily (qd) and two tablets each comprising 125 mgof Compound 2 every 12 hours (q12h). In one embodiment, 600 mg ofCompound 1 may be administered daily (qd) and 250 mg of Compound 2administered every 12 hours (q12h) by administering three tablets eachcomprising 200 mg of Compound 1 Form I, and 83.3 mg of substantiallyamorphous Compound 2 daily (qd) and two tablets each comprising 125 mgof Compound 2 every 12 hours (q12h).

These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein. In another aspect, the present invention featuresa kit comprising a pharmaceutical composition or tablet of the presentinvention comprising Compound 1 Form I and a solid dispersion comprisingsubstantially amorphous Compound 2, and a separate additionaltherapeutic agent or pharmaceutical composition thereof. In anotherembodiment, the pharmaceutical composition or tablet of the presentinvention, separate additional therapeutic agent or pharmaceuticalcomposition thereof are in separate containers. In another embodiment,the separate containers are bottles. In another embodiment, the separatecontainers are vials. In another embodiment, the separate containers areblister packs.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

Therapeutic Uses of the Composition

In one aspect, the invention also provides a method of treating,lessening the severity of, or symptomatically treating a disease in apatient, the method comprising administering an effective amount of thepharmaceutical composition or tablet of the invention to the patient,preferably a mammal, wherein the disease is selected from cysticfibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, such as protein C deficiency,Type 1 hereditary angioedema, lipid processing deficiencies, such asfamilial hypercholesterolemia, Type 1 chylomicronemia,abetalipoproteinemia, lysosomal storage diseases, such as I-celldisease/pseudo-Hurler, mucopolysaccharidoses, Sandhof/Tay-Sachs,Crigler-Najjar type II, polyendocrinopathy/hyperinsulemia, Diabetesmellitus, Laron dwarfism, myleoperoxidase deficiency, primaryhypoparathyroidism, melanoma, glycanosis CDG type 1, congenitalhyperthyroidism, osteogenesis imperfecta, hereditary hypofibrinogenemia,ACT deficiency, Diabetes insipidus (DI), neurophyseal DI, neprogenic DI,Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease,neurodegenerative diseases such as Alzheimer's disease, Parkinson'sdisease, amyotrophic lateral sclerosis, progressive supranuclear plasy,Pick's disease, several polyglutamine neurological disorders such asHuntington's, spinocerebullar ataxia type I, spinal and bulbar muscularatrophy, dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, COPD, dry-eye disease, or Sjogren'sdisease, osteoporosis, osteopenia, bone healing and bone growth(including bone repair, bone regeneration, reducing bone resorption andincreasing bone deposition), Gorham's Syndrome, chloride channelopathiessuch as myotonia congenita (Thomson and Becker forms), Bartter'ssyndrome type III, Dent's disease, hyperekplexia, epilepsy, lysosomalstorage disease, Angelman syndrome, and Primary Ciliary Dyskinesia(PCD), a term for inherited disorders of the structure and/or functionof cilia, including PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus and ciliary aplasia.

In one aspect, the invention also provides a method of treating,lessening the severity of, or symptomatically treating a disease in apatient comprising administering an effective amount of thepharmaceutical composition or tablet of the invention to the patient,preferably a mammal, wherein the disease is selected from generalizedepilepsy with ferbrile seizures plus (GEFS+), general epilepsy withferbile and aferbrile seizures, myotonia, paramyotonia congenital,potassium-aggravated myotonia, hyperkalemic periodic paralysis, LQTS,LQTS/Brugada syndrome, autosomal-dominant LQTS with deafness,autosomal-recessive LQTS, LQTS with dysmorphic features, congenital andacquired LQTS, Timothy syndrome, persistent hyperinsulinemichypolglycemia of infancy, dilated cardiomyopathy, autosomal-dominantLQTS, Dent disease, Osteopetrosis, Bartter syndrome type III, centralcore disease, malignant hyperthermia, and catecholaminergic polymorphictachycardia.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation N1303K, ΔI507, or R560T.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation G551D. In another embodiment, the patient is homozygousin G551D. In another embodiment, the patient is heterozygous in G551Dwherein the other CFTR genetic mutation is any one of ΔF508, G542X,N1303K, W1282X, R117H, R553X, 1717-1G→A, 621+1G→T, 2789+5G→A,3849+10kbC→T, R1162X, G85E, 3120+1G→A, ΔI507, 1898+1G→A, 3659delC,R347P, R560T, R334W, A455E, 2184delA, or 711+1G→T.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation ΔF508. In another embodiment, the patient is homozygousin ΔF508. In another embodiment, the patient is heterozygous in ΔF508wherein the other CFTR genetic mutation is any one of G551D, G542X,N1303K, W1282X, R117H, R553X, 1717-1G→A, 621+1G→T, 2789+5G→A,3849+10kbC→T, R1162X, G85E, 3120+1G→A, ΔI507, 1898+1G→A, 3659delC,R347P, R560T, R334W, A455E, 2184delA, or 71 l+1G→T.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, D110H,R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,F1074L, D110E, D1270N, D1152H, 1717-1G→A, 621+1G→T, 3120+1G→A, 1898+1G→A, 711+1 G→T, 2622+1 G→A, 405+1 G→A, 406-1 G→A, 4005+1 G→A, 1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A, 4374+1G→T,3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A, 3120G→A,1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C, 405+3A→C,1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A, 1898+1G→T,4005+2T→C and 621+3A→G.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V and G1069R. In oneembodiment of this aspect, the invention provides a method of treatingCFTR comprising administering Compound 1 to a patient possessing a humanCFTR mutation selected from G178R, G551S, G970R, G1244E, S1255P, G1349D,S549N, S549R and S1251N. In one aspect, the present invention isdirected to a method of treating, lessening the severity of, orsymptomatically treating cystic fibrosis in a patient comprisingadministering an effective amount of the pharmaceutical composition ortablet of the invention to the patient, preferably a mammal, wherein thepatient possesses the CFTR genetic mutation is selected from E193K,F1052V and G1069R. In some embodiments of this aspect, the methodproduces a greater than 10-fold increase in chloride transport relativeto baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from R117C, D110H, R347H, R352Q, E56K,P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270Nand D1152H. In one embodiment of this aspect, the method produces anincrease in chloride transport which is greater or equal to 10% abovethe baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from 1717-1G→A, 621+1G→T, 3120+1G→A,1898+1G→A, 711+1G→T, 2622+1G→A, 405+1G→A, 406-1G→A, 4005+1G→A,1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A,4374+1G→T, 3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A,3120G→A, 1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C,405+3A→C, 1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A,1898+1G→T, 4005+2T→C and 621+3A→G. In one aspect, the present inventionis directed to a method of treating, lessening the severity of, orsymptomatically treating cystic fibrosis in a patient comprisingadministering an effective amount of the pharmaceutical composition ortablet of the invention to the patient, preferably a mammal, wherein thepatient possesses the CFTR genetic mutation is selected from 1717-1 G→A,1811+1.6kbA→G, 2789+5G→A, 3272-26A→G and 3849+10kbC→T. In one aspect,the present invention is directed to a method of treating, lessening theseverity of, or symptomatically treating cystic fibrosis in a patientcomprising administering an effective amount of the pharmaceuticalcomposition or tablet of the invention to the patient, preferably amammal, wherein the patient possesses the CFTR genetic mutation isselected from 2789+5G→A and 3272-26A→G.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, D110H,R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,F1074L, D110E, D1270N, D1152H, 1717-1G→A, 621+1G→T, 3120+1G→A, 1898+1G→A, 711+1 G→T, 2622+1 G→A, 405+1 G→A, 406-1 G→A, 4005+1 G→A, 1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A, 4374+1G→T,3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A, 3120G→A,1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C, 405+3A→C,1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A, 1898+1G→T,4005+2T→C and 621+3A→G, and a human CFTR mutation selected from ΔF508,R117H, and G551D.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V and G1069R, and a human CFTRmutation selected from ΔF508, R117H, and G551D. In one aspect, thepresent invention is directed to a method of treating, lessening theseverity of, or symptomatically treating cystic fibrosis in a patientcomprising administering an effective amount of the pharmaceuticalcomposition or tablet of the invention to the patient, preferably amammal, wherein the patient possesses the CFTR genetic mutation isselected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549Rand S1251N, and a human CFTR mutation selected from ΔF508, R117H, andG551D. In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from E193K, F1052V and G1069R, and a humanCFTR mutation selected from ΔF508, R117H, and G551D. In some embodimentsof this aspect, the method produces a greater than 10-fold increase inchloride transport relative to baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from R117C, D110H, R347H, R352Q, E56K,P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270Nand D1152H, and a human CFTR mutation selected from ΔF508, R117H, andG551D. In one embodiment of this aspect, the method produces an increasein chloride transport which is greater or equal to 10% above thebaseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from 1717-1G→A, 621+1G→T, 3120+1G→A,1898+1G→A, 711+1G→T, 2622+1G→A, 405+1G→A, 406-1 G→A, 4005+1 G→A, 1812-1G→A, 1525-1 G→A, 712-1 G→T, 1248+1 G→A, 1341+1G→A, 3121-1G→A, 4374+1G→T,3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A, 3120G→A,1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C, 405+3A→C,1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A, 1898+1G→T,4005+2T→C and 621+3A→G, and a human CFTR mutation selected from ΔF508,R117H, and G551D. In one aspect, the present invention is directed to amethod of treating, lessening the severity of, or symptomaticallytreating cystic fibrosis in a patient comprising administering aneffective amount of the pharmaceutical composition or tablet of theinvention to the patient, preferably a mammal, wherein the patientpossesses the CFTR genetic mutation is selected from 1717-1G→A,1811+1.6kbA→G, 2789+5G→A, 3272-26A→G and 3849+10kbC→T, and a human CFTRmutation selected from ΔF508, R117H, and G551D. In one aspect, thepresent invention is directed to a method of treating, lessening theseverity of, or symptomatically treating cystic fibrosis in a patientcomprising administering an effective amount of the pharmaceuticalcomposition or tablet of the invention to the patient, preferably amammal, wherein the patient possesses the CFTR genetic mutation isselected from 2789+5G→A and 3272-26A→G, and a human CFTR mutationselected from ΔF508, R117H.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, D10H, R347H,R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L,D110E, D1270N, D1152H, 1717-1G→A, 621+1G→T, 3120+1G→A, 1898+1G→A,711+1G→T, 2622+1G→A, 405+1G→A, 406-1G→A, 4005+1G→A, 1812-1G→A,1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A, 4374+1G→T,3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A, 3120G→A,1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C, 405+3A→C,1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A, 1898+1G→T,4005+2T→C and 621+3A→G, and a human CFTR mutation selected from ΔF508,R117H, and G551D.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V and G1069R. In one aspect,the present invention is directed to a method of treating, lessening theseverity of, or symptomatically treating cystic fibrosis in a patientcomprising administering an effective amount of the pharmaceuticalcomposition or tablet of the invention to the patient, preferably amammal, wherein the patient possesses the CFTR genetic mutation isselected from G178R, G551S, G970R, G1244E, S1255P, G1349D, S549N, S549Rand S1251N. In one aspect, the present invention is directed to a methodof treating, lessening the severity of, or symptomatically treatingcystic fibrosis in a patient comprising administering an effectiveamount of the pharmaceutical composition or tablet of the invention tothe patient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from E193K, F1052V and G1069R. In someembodiments of this aspect, the method produces a greater than 10-foldincrease in chloride transport relative to baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from R117C, D110H, R347H, R352Q, E56K,P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270Nand D1152H. In one embodiment of this aspect, the method produces anincrease in chloride transport which is greater or equal to 10% abovethe baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from 1717-1G→A, 621+1G→T, 3120+1G→A,1898+1G→A, 711+1G→T, 2622+1G→A, 405+1G→A, 406-1G→A, 4005+1G→A,1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A,4374+1G→T, 3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A,3120G→A, 1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C,405+3A→C, 1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A,1898+1G→T, 4005+2T→C and 621+3A→G. In one aspect, the present inventionis directed to a method of treating, lessening the severity of, orsymptomatically treating cystic fibrosis in a patient comprisingadministering an effective amount of the pharmaceutical composition ortablet of the invention to the patient, preferably a mammal, wherein thepatient possesses the CFTR genetic mutation is selected from 1717-1G→A,1811+1.6kbA→G, 2789+5G→A, 3272-26A→G and 3849+10kbC→T. In one aspect,the present invention is directed to a method of treating, lessening theseverity of, or symptomatically treating cystic fibrosis in a patientcomprising administering an effective amount of the pharmaceuticalcomposition or tablet of the invention to the patient, preferably amammal, wherein the patient possesses the CFTR genetic mutation isselected from 2789+5G→A and 3272-26A→G.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V, G1069R, R117C, D110H,R347H, R352Q, E56K, P67L, L206W, A455E, D579G, S1235R, S945L, R1070W,F1074L, D110E, D1270N, D1152H, 1717-1G→A, 621+1G→T, 3120+1G→A,1898+1G→A, 711+1G→T, 2622+1G→A, 405+1G→A, 406-1G→A, 4005+1G→A,1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A,4374+1G→T, 3850-1 G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A,3120G→A, 1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C,405+3A→C, 1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A,1898+1G→T, 4005+2T→C and 621+3A→G, and a human CFTR mutation selectedfrom ΔF508, R117H, and G551D, and one or more human CFTR mutationsselected from ΔF508, R117H, and G551D.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from G178R, G551S, G970R, G1244E, S1255P,G1349D, S549N, S549R, S1251N, E193K, F1052V and G1069R, and one or morehuman CFTR mutations selected from ΔF508, RI 17H, and G551D. In oneaspect, the present invention is directed to a method of treating,lessening the severity of, or symptomatically treating cystic fibrosisin a patient comprising administering an effective amount of thepharmaceutical composition or tablet of the invention to the patient,preferably a mammal, wherein the patient possesses the CFTR geneticmutation is selected from G178R, G551S, G970R, G1244E, S1255P, G1349D,S549N, S549R and S1251N, and one or more human CFTR mutations selectedfrom ΔF508, R117H, and G551D. In one aspect, the present invention isdirected to a method of treating, lessening the severity of, orsymptomatically treating cystic fibrosis in a patient comprisingadministering an effective amount of the pharmaceutical composition ortablet of the invention to the patient, preferably a mammal, wherein thepatient possesses the CFTR genetic mutation is selected from E193K,F1052V and G1069R, and one or more human CFTR mutations selected fromΔF508, R117H, and G551D. In some embodiments of this aspect, the methodproduces a greater than 10-fold increase in chloride transport relativeto baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from R117C, D110H, R347H, R352Q, E56K,P67L, L206W, A455E, D579G, S1235R, S945L, R1070W, F1074L, D110E, D1270Nand D1152H, and one or more human CFTR mutations selected from ΔF508,R117H, and G551D. In one embodiment of this aspect, the method producesan increase in chloride transport which is greater or equal to 10% abovethe baseline chloride transport.

In one aspect, the present invention is directed to a method oftreating, lessening the severity of, or symptomatically treating cysticfibrosis in a patient comprising administering an effective amount ofthe pharmaceutical composition or tablet of the invention to thepatient, preferably a mammal, wherein the patient possesses the CFTRgenetic mutation is selected from 1717-1G→A, 621+1G→T, 3120+1G→A,1898+1G→A, 711+1G→T, 2622+1G→A, 405+1G→A, 406-1G→A, 4005+1G→A,1812-1G→A, 1525-1G→A, 712-1G→T, 1248+1G→A, 1341+1G→A, 3121-1G→A,4374+1G→T, 3850-1G→A, 2789+5G→A, 3849+10kbC→T, 3272-26A→G, 711+5G→A,3120G→A, 1811+1.6kbA→G, 711+3A→G, 1898+3A→G, 1717-8G→A, 1342-2A→C,405+3A→C, 1716G/A, 1811+1G→C, 1898+5G→T, 3850-3T→G, IVS14b+5G→A,1898+1G→T, 4005+2T→C and 621+3A→G, and one or more human CFTR mutationsselected from ΔF508, R117H, and G551D. In one aspect, the presentinvention is directed to a method of treating, lessening the severityof, or symptomatically treating cystic fibrosis in a patient comprisingadministering an effective amount of the pharmaceutical composition ortablet of the invention to the patient, preferably a mammal, wherein thepatient possesses the CFTR genetic mutation is selected from 1717-1G→A,1811+1.6kbA→G, 2789+5G→A, 3272-26A→G and 3849+10kbC→T, and one or morehuman CFTR mutations selected from ΔF508, R117H, and G551D. In oneaspect, the present invention is directed to a method of treating,lessening the severity of, or symptomatically treating cystic fibrosisin a patient comprising administering an effective amount of thepharmaceutical composition or tablet of the invention to the patient,preferably a mammal, wherein the patient possesses the CFTR geneticmutation is selected from 2789+5G→A and 3272-26A→G, and one or morehuman CFTR mutations selected from ΔF508, R117H, and G551D.

In certain embodiments, the pharmaceutically acceptable composition ortablet of the present invention comprising Compound 1 Form I and a soliddispersion of substantially amorphous Compound 2 are useful fortreating, lessening the severity of, or symptomatically treating cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508, as well as other mutations such as the G551Dmutation, or the R117H mutation. In certain embodiments, thepharmaceutically acceptable compositions or tablets comprising Compound1 Form I and a solid dispersion comprising substantially amorphousCompound 2 are useful for treating, lessening the severity of, orsymptomatically treating cystic fibrosis in patients who exhibit littleto no residual CFTR activity. In certain embodiments, thepharmaceutically acceptable compositions or tablets comprising Compound1 Form I and a solid dispersion comprising substantially amorphousCompound 2 are useful for treating, lessening the severity of, orsymptomatically treating cystic fibrosis in patients who exhibit littleto no residual CFTR activity in the apical membrane of respiratoryepithelia.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods. In another embodiment, thecompounds and compositions of the present invention are useful fortreating or lessening the severity of cystic fibrosis in patients whohave residual CFTR activity induced or augmented using or gene therapy.Such methods increase the amount of CFTR present at the cell surface,thereby inducing a hitherto absent CFTR activity in a patient oraugmenting the existing level of residual CFTR activity in a patient.

In one embodiment, pharmaceutical compositions and tablets of thepresent invention comprising Compound 1 Form I and a solid dispersioncomprising substantially amorphous Compound 2, as described herein, areuseful for treating or lessening the severity of cystic fibrosis inpatients within certain genotypes exhibiting residual CFTR activity,e.g., Class I mutations (not synthesized), class II mutation(misfolding), class III mutations (impaired regulation or gating), classIV mutations (altered conductance), or class V mutations (reducedsynthesis).

In one embodiment, pharmaceutical compositions and tablets of thepresent invention comprising Compound 1 Form I and a solid dispersioncomprising substantially amorphous Compound 2, as described herein, areuseful for treating, lessening the severity of, or symptomaticallytreating cystic fibrosis in patients within certain clinical phenotypes,e.g., a moderate to mild clinical phenotype that typically correlateswith the amount of residual CFTR activity in the apical membrane ofepithelia. Such phenotypes include patients exhibiting pancreaticsufficiency.

In one embodiment, pharmaceutical compositions and tablets of thepresent invention comprising Compound 1 Form I and a solid dispersioncomprising substantially amorphous Compound 2, as described herein, areuseful for treating, lessening the severity of, or symptomaticallytreating patients diagnosed with pancreatic sufficiency, idiopathicpancreatitis and congenital bilateral absence of the vas deferens, ormild lung disease wherein the patient exhibits residual CFTR activity.

In one embodiment, pharmaceutical compositions and tablets of thepresent invention comprising Compound 1 Form I and a solid dispersioncomprising substantially amorphous Compound 2, as described herein, areuseful for treating, lessening the severity of, or symptomaticallytreating patients diagnosed with pancreatic sufficiency, idiopathicpancreatitis and congenital bilateral absence of the vas deferens, ormild lung disease wherein the patient has wild type CFTR.

In addition to cystic fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. These include, but are not limited to, chronic obstructivepulmonary disease (COPD), dry eye disease, and Sjögren's Syndrome. COPDis characterized by airflow limitation that is progressive and not fullyreversible. The airflow limitation is due to mucus hypersecretion,emphysema, and bronchiolitis. Activators of mutant or wild-type CFTRoffer a potential treatment of mucus hypersecretion and impairedmucociliary clearance that is common in COPD. Specifically, increasinganion secretion across CFTR may facilitate fluid transport into theairway surface liquid to hydrate the mucus and optimized periciliaryfluid viscosity. This would lead to enhanced mucociliary clearance and areduction in the symptoms associated with COPD. Dry eye disease ischaracterized by a decrease in tear aqueous production and abnormal tearfilm lipid, protein and mucin profiles. There are many causes of dryeye, some of which include age, Lasik eye surgery, arthritis,medications, chemical/thermal burns, allergies, and diseases, such ascystic fibrosis and Sjögrens's syndrome. Increasing anion secretion viaCFTR would enhance fluid transport from the corneal endothelial cellsand secretory glands surrounding the eye to increase corneal hydration.This would help to alleviate the symptoms associated with dry eyedisease. Sjögrens's syndrome is an autoimmune disease in which theimmune system attacks moisture-producing glands throughout the body,including the eye, mouth, skin, respiratory tissue, liver, vagina, andgut. Symptoms, include, dry eye, mouth, and vagina, as well as lungdisease. The disease is also associated with rheumatoid arthritis,systemic lupus, systemic sclerosis, and polymypositis/dermatomyositis.Defective protein trafficking is believed to cause the disease, forwhich treatment options are limited. Augmenters or inducers of CFTRactivity may hydrate the various organs afflicted by the disease andhelp to elevate the associated symptoms.

In one embodiment, the invention relates to a method of augmenting orinducing anion channel activity in vitro or in vivo, comprisingcontacting the channel with any one of pharmaceutical compositions PC-Ito PC-XXV. In another embodiment, the anion channel is a chloridechannel or a bicarbonate channel. In another embodiment, the anionchannel is a chloride channel.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the invention will be decided by the attendingphysician within the scope of sound medical judgment. The specificeffective dose level for any particular patient or organism will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed, and like factors wellknown in the medical arts. The term “patient”, as used herein, means ananimal, preferably a mammal, and most preferably a human.

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

Examples XRPD (X-Ray Powder Diffraction)

The X-Ray diffraction (XRD) data of Compound 1 Form I were collected ona Bruker D8 DISCOVER powder diffractometer with HI-STAR 2-dimensionaldetector and a flat graphite monochromator. Cu sealed tube with Kαradiation was used at 40 kV, 35 mA. The samples were placed onzero-background silicon wafers at 25° C. For each sample, two dataframes were collected at 120 seconds each at 2 different θ₂ angles: 8°and 260. The data were integrated with GADDS software and merged withDIFFRACT^(plus)EVA software. Uncertainties for the reported peakpositions are ±0.2 degrees.

Differential Scanning Calorimetry (DSC)

The Differential scanning calorimetry (DSC) data of Compound 1 Form Iwere collected using a DSC Q100 V9.6 Build 290 (TA Instruments, NewCastle, Del.). Temperature was calibrated with indium and heat capacitywas calibrated with sapphire. Samples of 3-6 mg were weighed intoaluminum pans that were crimped using lids with 1 pin hole. The sampleswere scanned from 25° C. to 350° C. at a heating rate of 1.0° C./min andwith a nitrogen gas purge of 50 ml/min. Data were collected by ThermalAdvantage Q Series™ version 2.2.0.248 software and analyzed by UniversalAnalysis software version 4.1D (TA Instruments, New Castle, Del.). Thereported numbers represent single analyses.

Compound 1 Form I Single Crystal Structure Determination

Diffraction data were acquired on Bruker Apex II diffractometer equippedwith sealed tube Cu K-alpha source and an Apex II CCD detector. Thestructure was solved and refined using SHELX program (Sheldrick, G. M.,Acta Cryst., (2008) A64, 112-122). Based on systemratic absences andintensities statistics the structure was solved and refined in P2₁/nspace group.

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals.

2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchased fromSaltigo (an affiliate of the Lanxess Corporation).

Preparation of Compound 1 Preparation of(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) was slurried in toluene (10 vol). Vitride® (2 eq) was added viaaddition funnel at a rate to maintain the temperature at 15-25° C. Atthe end of the addition, the temperature was increased to 40° C. for 2hours (h), then 10% (w/w) aqueous (aq) NaOH (4.0 eq) was carefully addedvia addition funnel, maintaining the temperature at 40-50° C. Afterstirring for an additional 30 minutes (min), the layers were allowed toseparate at 40° C. The organic phase was cooled to 20° C., then washedwith water (2×1.5 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that was useddirectly in the next step.

Preparation of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) was dissolved inMTBE (5 vol). A catalytic amount of 4-(N,N-dimethyl)aminopyridine (DMAP)(1 mol %) was added and SOCl₂ (1.2 eq) was added via addition funnel.The SOCl₂ was added at a rate to maintain the temperature in the reactorat 15-25° C. The temperature was increased to 30° C. for 1 h, and thenwas cooled to 20° C. Water (4 vol) was added via addition funnel whilemaintaining the temperature at less than 30° C. After stirring for anadditional 30 min, the layers were allowed to separate. The organiclayer was stirred and 10% (w/v) aq NaOH (4.4 vol) was added. Afterstirring for 15 to 20 min, the layers were allowed to separate. Theorganic phase was then dried (Na₂SO₄), filtered, and concentrated toafford crude 5-chloromethyl-2,2-difluoro-1,3-benzodioxole that was useddirectly in the next step.

Preparation of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) was added to a slurry of NaCN (1.4 eq) in DMSO (3 vol),while maintaining the temperature between 30-40° C. The mixture wasstirred for 1 h, and then water (6 vol) was added, followed by methyltert-butyl ether (MTBE) (4 vol). After stirring for 30 min, the layerswere separated. The aqueous layer was extracted with MTBE (1.8 vol). Thecombined organic layers were washed with water (1.8 vol), dried(Na₂SO₄), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) that was useddirectly in the next step.

Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile

A reactor was purged with nitrogen and charged with 900 mL of toluene.The solvent was degassed via nitrogen sparge for no less than 16 h. Tothe reactor was then charged Na₃PO₄ (155.7 g, 949.5 mmol), followed bybis(dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A 10% w/wsolution of tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) wascharged over 10 min at 23° C. from a nitrogen purged addition funnel.The mixture was allowed to stir for 50 min, at which time5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added over1 min. After stirring for an additional 50 min, the mixture was chargedwith ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 min followed bywater (4.5 mL) in one portion. The mixture was heated to 70° C. over 40min and analyzed by HPLC every 1-2 h for the percent conversion of thereactant to the product. After complete conversion was observed(typically 100% conversion after 5-8 h), the mixture was cooled to20-25° C. and filtered through a celite pad. The celite pad was rinsedwith toluene (2×450 mL) and the combined organics were concentrated to300 mL under vacuum at 60-65° C. The concentrate was charged with 225 mLDMSO and concentrated under vacuum at 70-80° C. until activedistillation of the solvent ceased. The solution was cooled to 20-25° C.and diluted to 900 mL with DMSO in preparation for Step 2. ¹H NMR (500MHz, CDCl₃) δ 7.16-7.10 (m, 2H), 7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H),4.19 (m, 2H), 1.23 (t, J=7.1 Hz, 3H).

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

The DMSO solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile fromabove was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20 min whilemaintaining an internal temperature <40° C. The mixture was then heatedto 75° C. over 1 h and analyzed by HPLC every 1-2 h for % conversion.When a conversion of >99% was observed (typically after 5-6 h), thereaction was cooled to 20-25° C. and extracted with MTBE (2×525 mL),with sufficient time to allow for complete phase separation during theextractions. The combined organic extracts were washed with 5% NaCl(2×375 mL). The solution was then transferred to equipment appropriatefor a 1.5-2.5 Torr vacuum distillation that was equipped with a cooledreceiver flask. The solution was concentrated under vacuum at <60° C. toremove the solvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrilewas then distilled from the resulting oil at 125-130° C. (oventemperature) and 1.5-2.0 Torr.(2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was isolated as a clearoil in 66% yield from 5-bromo-2,2-difluoro-1,3-benzodioxole (2 steps)and with an HPLC purity of 91.5% AUC (corresponds to a w/w assay of95%). ¹H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J=8.4 Hz, 1H),7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s, 2H).

Preparation of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile

A mixture of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (1.0 eq),50 wt % aqueous KOH (5.0 eq) 1-bromo-2-chloroethane (1.5 eq), andOct₄NBr (0.02 eq) was heated at 70° C. for 1 h. The reaction mixture wascooled, then worked up with MTBE and water. The organic phase was washedwith water and brine. The solvent was removed to afford(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic Acid

(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile washydrolyzed using 6 M NaOH (8 equiv) in ethanol (5 vol) at 80° C.overnight. The mixture was cooled to room temperature and the ethanolwas evaporated under vacuum. The residue was taken up in water and MTBE,1 M HCl was added, and the layers were separated. The MTBE layer wasthen treated with dicyclohexylamine (DCHA) (0.97 equiv). The slurry wascooled to 0° C., filtered and washed with heptane to give thecorresponding DCHA salt. The salt was taken into MTBE and 10% citricacid and stirred until all the solids had dissolved. The layers wereseparated and the MTBE layer was washed with water and brine. A solventswap to heptane followed by filtration gave1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid afterdrying in a vacuum oven at 50° C. overnight.

Preparation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonyl chloride

1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.2eq) is slurried in toluene (2.5 vol) and the mixture was heated to 60°C. SOCl₂ (1.4 eq) was added via addition funnel. The toluene and SOCl₂were distilled from the reaction mixture after 30 minutes. Additionaltoluene (2.5 vol) was added and the resulting mixture was distilledagain, leaving the product acid chloride as an oil, which was usedwithout further purification.

Preparation of tert-butyl-3-(3-methylpyridin-2-yl)benzoate

2-Bromo-3-methylpyridine (1.0 eq) was dissolved in toluene (12 vol).K₂CO₃ (4.8 eq) was added, followed by water (3.5 vol). The resultingmixture was heated to 65° C. under a stream of N₂ for 1 hour.3-(t-Butoxycarbonyl)phenylboronic acid (1.05 eq) and Pd(dppf)Cl₂—CH₂Cl₂(0.015 eq) were then added and the mixture was heated to 80° C. After 2hours, the heat was turned off, water was added (3.5 vol), and thelayers were allowed to separate. The organic phase was then washed withwater (3.5 vol) and extracted with 10% aqueous methanesulfonic acid (2eq MsOH, 7.7 vol). The aqueous phase was made basic with 50% aqueousNaOH (2 eq) and extracted with EtOAc (8 vol). The organic layer wasconcentrated to afford crude tert-butyl-3-(3-methylpyridin-2-yl)benzoate(82%) that was used directly in the next step.

Preparation of2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide

tert-Butyl-3-(3-methylpyridin-2-yl)benzoate (1.0 eq) was dissolved inEtOAc (6 vol). Water (0.3 vol) was added, followed by urea-hydrogenperoxide (3 eq). Phthalic anhydride (3 eq) was then added portionwise tothe mixture as a solid at a rate to maintain the temperature in thereactor below 45° C. After completion of the phthalic anhydrideaddition, the mixture was heated to 45° C. After stirring for anadditional 4 hours, the heat was turned off. 10% w/w aqueous Na₂SO₃ (1.5eq) was added via addition funnel. After completion of Na₂SO₃ addition,the mixture was stirred for an additional 30 min and the layersseparated. The organic layer was stirred and 10% wt/wt aqueous. Na₂CO₃(2 eq) was added. After stirring for 30 minutes, the layers were allowedto separate. The organic phase was washed 13% w/v aq NaCl. The organicphase was then filtered and concentrated to afford crude2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide (95%) thatwas used directly in the next step.

Preparation of tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate

A solution of 2-(3-(tert-butoxycarbonyl)phenyl)-3-methylpyridine-1-oxide(1 eq) and pyridine (4 eq) in acetonitrile (8 vol) was heated to 70° C.A solution of methanesulfonic anhydride (1.5 eq) in MeCN (2 vol) wasadded over 50 min via addition funnel while maintaining the temperatureat less than 75° C. The mixture was stirred for an additional 0.5 hoursafter complete addition. The mixture was then allowed to cool toambient. Ethanolamine (10 eq) was added via addition funnel. Afterstirring for 2 hours, water (6 vol) was added and the mixture was cooledto 10° C. After stirring for 3 hours, the solid was collected byfiltration and washed with water (3 vol), 2:1 acetonitrile/water (3vol), and acetonitrile (2×1.5 vol). The solid was dried to constantweight (<1% difference) in a vacuum oven at 50° C. with a slight N₂bleed to afford tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate as ared-yellow solid (53% yield).

Preparation of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate

The crude acid chloride described above was dissolved in toluene (2.5vol based on acid chloride) and added via addition funnel to a mixtureof tert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate (1 eq), DMAP,(0.02 eq), and triethylamine (3.0 eq) in toluene (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate). After 2 hours,water (4 vol based ontert-butyl-3-(6-amino-3-methylpyridin-2-yl)benzoate) was added to thereaction mixture. After stirring for 30 minutes, the layers wereseparated. The organic phase was then filtered and concentrated toafford a thick oil of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate(quantitative crude yield). Acetonitrile (3 vol based on crude product)was added and distilled until crystallization occurs. Water (2 vol basedon crude product) was added and the mixture stirred for 2 h. The solidwas collected by filtration, washed with 1:1 (by volume)acetonitrile/water (2×1 volumes based on crude product), and partiallydried on the filter under vacuum. The solid was dried to a constantweight (<1% difference) in a vacuum oven at 60° C. with a slight N₂bleed to afford 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate as abrown solid.

Preparation of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCL Salt

To a slurry of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq)in MeCN (3.0 vol) was added water (0.83 vol) followed by concentratedaqueous HCl (0.83 vol). The mixture was heated to 45+5° C. Afterstirring for 24 to 48 h, the reaction was complete, and the mixture wasallowed to cool to ambient. Water (1.33 vol) was added and the mixturestirred. The solid was collected by filtration, washed with water (2×0.3vol), and partially dried on the filter under vacuum. The solid wasdried to a constant weight (<1% difference) in a vacuum oven at 60° C.with a slight N₂ bleed to afford3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCl as anoff-white solid.

An ¹HNMR spectrum of Compound 1 is shown in FIG. 8 and FIG. 9 depicts an¹HNMR spectrum of Compound 1 as an HCl salt.

Table 2 below recites the ¹HNMR data for Compound 1.

TABLE 2 Compound LC/MS LC/RT No M + 1 minutes NMR 1 453.3 1.93 ¹HNMR(400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99- 7.93 (m, 3H), 7.80-7.78 (m, 1H),7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m, 2H), 2.24 (s, 3H),1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

Preparation of Compound 1 Form I Preparation of Compound 1 Form I,Method A

A slurry of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoic acid.HCl (1 eq) inwater (10 vol) was stirred at ambient temperature. A sample was takenafter stirring for 24 h. The sample was filtered and the solid waswashed with water (2 times). The solid sample was submitted for DSCanalysis. When DSC analysis indicated complete conversion to Form I, thesolid was collected by filtration, washed with water (2×1.0 vol), andpartially dried on a filter under vacuum. The solid was then dried to aconstant weight (<1% difference) in a vacuum oven at 60° C. with aslight N₂ bleed to afford Compound 1 Form I as an off-white solid (98%yield). ¹H NMR (400 MHz, DMSO-d6) 9.14 (s, 1H), 7.99-7.93 (m, 3H),7.80-7.78 (m, 1H), 7.74-7.72 (m, 1H), 7.60-7.55 (m, 2H), 7.41-7.33 (m,2H), 2.24 (s, 3H), 1.53-1.51 (m, 2H), 1.19-1.17 (m, 2H).

Preparation of Compound 1 Form I, Method B

A solution of 3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate (1.0 eq)in formic acid (3.0 vol) was heated with stirring to 70±10° C., for 8 h.The reaction was deemed complete when no more than 1.0% AUC bychromatographic methods of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate)remained. The mixture was allowed to cool to ambient. The solution wasadded to water (6 vol), heated at 50° C., and the mixture was stirred.The mixture was then heated to 70±10° C. until the level of3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)-t-butylbenzoate was nomore than 0.8% (AUC). The solid was collected by filtration, washed withwater (2×3 vol), and partially dried on the filter under vacuum. Thesolid was dried to a constant weight (<1% difference) in a vacuum ovenat 60° C. with a slight N₂ bleed to afford Compound 1 Form I as anoff-white solid.

The DSC trace of Compound 1 Form I is shown in FIG. 10. Melting forCompound 1 Form I occurs at about 204° C.

An X-ray diffraction pattern was calculated from a single crystalstructure of Compound 1 Form I and is shown in FIG. 1. Table 3 lists thecalculated peaks for FIG. 1.

TABLE 3 2θ Angle Relative Peak Rank [degrees] Intensity [%] 11 14.4148.2 8 14.64 58.8 1 15.23 100.0 2 16.11 94.7 3 17.67 81.9 7 19.32 61.3 421.67 76.5 5 23.40 68.7 9 23.99 50.8 6 26.10 67.4 10 28.54 50.1

An actual X-ray powder diffraction pattern of Compound 1 Form I is shownin FIG. 2. Table 4 lists the actual peaks for FIG. 2.

TABLE 4 2θ Angle Relative Peak Rank [degrees] Intensity [%] 7 7.83 37.73 14.51 74.9 4 14.78 73.5 1 15.39 100.0 2 16.26 75.6 6 16.62 42.6 517.81 70.9 9 21.59 36.6 10 23.32 34.8 11 24.93 26.4 8 25.99 36.9

Colorless crystals of Compound 1 Form I were obtained by cooling aconcentrated 1-butanol solution from 75° C. to 10° C. at a rate of 0.2°C./min. A crystal with dimensions of 0.50×0.08×0.03 mm was selected,cleaned with mineral oil, mounted on a MicroMount and centered on aBruker APEX II system. Three batches of 40 frames separated inreciprocal space were obtained to provide an orientation matrix andinitial cell parameters. Final cell parameters were obtained and refinedbased on the full data set.

A diffraction data set of reciprocal space was obtained to a resolutionof 0.82 Å using 0.5° steps using 30 s exposure for each frame. Data werecollected at 100 (2) K. Integration of intensities and refinement ofcell parameters were accomplished using APEXII software. Observation ofthe crystal after data collection showed no signs of decomposition.

A conformational picture of Compound 1 Form I based on single crystalX-ray analysis is shown in FIG. 11. Compound 1 Form I is monoclinic,P₂1/n, with the following unit cell dimensions: a=4.9626(7) Å,b=12.299(2) Å, c=33.075 (4) Å, β=93.938(9) °, V=2014.0 Å³, Z=4. Densityof Compound 1 Form I calculated from structural data is 1.492 g/cm³ at100 K.

Preparation of Compound 2 Synthesis of4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (26)

Procedure for the preparation of ethyl4-oxo-1,4-dihydroquinoline-3-carboxylate (25)

Compound 23 (4.77 g, 47.7 mmol) was added dropwise to compound 22 (10 g,46.3 mmol) with subsurface N₂ flow to drive out ethanol below 30° C. for0.5 hours. The solution was then heated to 100-110° C. and stirred for2.5 hours. After cooling the mixture to below 60° C., diphenyl ether wasadded. The resulting solution was added dropwise to diphenyl ether thathad been heated to 228-232° C. for 1.5 hours with subsurface N₂ flow todrive out ethanol. The mixture was stirred at 228-232° C. for another 2hours, cooled to below 100° C. and then heptane was added to precipitatethe product. The resulting slurry was stirred at 30° C. for 0.5 hours.The solids were then filtrated, and the cake was washed with heptane anddried in vacuo to give compound 25 as brown solid. ¹H NMR (DMSO-d₆; 400MHz) δ 12.25 (s), δ 8.49 (d), δ 8.10 (m), δ 7.64 (m), δ 7.55 (m), δ 7.34(m), δ 4.16 (q), δ 1.23 (t).

Procedure for the Preparation of 4-oxo-1,4-dihydroquinoline-3-carboxylicAcid (26)

Method 1

Compound 25 (1.0 eq) was suspended in a solution of HCl (10.0 eq) andH₂O (11.6 vol). The slurry was heated to 85-90° C., although alternativetemperatures are also suitable for this hydrolysis step. For example,the hydrolysis can alternatively be performed at a temperature of fromabout 75 to about 100° C. In some instances, the hydrolysis is performedat a temperature of from about 80 to about 95° C. In others, thehydrolysis step is performed at a temperature of from about 82 to about93° C. (e.g., from about 82.5 to about 92.5° C. or from about 86 toabout 89° C.). After stirring at 85-90° C. for approximately 6.5 hours,the reaction was sampled for reaction completion. Stirring may beperformed under any of the temperatures suited for the hydrolysis. Thesolution was then cooled to 20-25° C. and filtered. The reactor/cake wasrinsed with H₂O (2 vol×2). The cake was then washed with 2 vol H₂O untilthe pH ≥3.0. The cake was then dried under vacuum at 60° C. to givecompound 26.

Method 2

Compound 25 (11.3 g, 52 mmol) was added to a mixture of 10% NaOH (aq)(10 mL) and ethanol (100 mL). The solution was heated to reflux for 16hours, cooled to 20-25° C. and then the pH was adjusted to 2-3 with 8%HCl. The mixture was then stirred for 0.5 hours and filtered. The cakewas washed with water (50 mL) and then dried in vacuo to give compound26 as a brown solid. ¹H NMR (DMSO-d₆; 400 MHz) δ 15.33 (s), δ 13.39 (s),δ 8.87 (s), δ 8.26 (m), δ 7.87 (m), δ 7.80 (m), δ 7.56 (m).

Total Synthesis ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 2)

Procedure for the Preparation of 2,4-di-tert-butylphenyl methylcarbonate (30)

Method 1

To a solution of 2,4-di-tert-butyl phenol, 29, (10 g, 48.5 mmol) indiethyl ether (100 mL) and triethylamine (10.1 mL, 72.8 mmol), was addedmethyl chloroformate (7.46 mL, 97 mmol) dropwise at 0° C. The mixturewas then allowed to warm to room temperature and stir for an additional2 hours. An additional 5 mL triethylamine and 3.7 mL methylchloroformate was then added and the reaction stirred overnight. Thereaction was then filtered, the filtrate was cooled to 0° C., and anadditional 5 mL triethylamine and 3.7 mL methyl chloroformate was thenadded and the reaction was allowed to warm to room temperature and thenstir for an addition 1 hours. At this stage, the reaction was almostcomplete and was worked up by filtering, then washing with water (2×),followed by brine. The solution was then concentrated to produce ayellow oil and purified using column chromatography to give compound 30.¹H NMR (400 MHz, DMSO-d₆) δ 7.35 (d, J=2.4 Hz, 1H), 7.29 (dd, J=8.4, 2.4Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 3.85 (s, 3H), 1.30 (s, 9H), 1.29 (s,9H).

Method 2

To a reactor vessel charged with 4-dimethylaminopyridine (DMAP, 3.16 g,25.7 mmol) and 2,4-ditert-butyl phenol (compound 29, 103.5 g, 501.6mmol) was added methylene chloride (415 g, 313 mL) and the solution wasagitated until all solids dissolved. Triethylamine (76 g, 751 mmol) wasthen added and the solution was cooled to 0-5° C. Methyl chloroformate(52 g, 550.3 mmol) was then added dropwise over 2.5-4 hours, whilekeeping the solution temperature between 0-5° C. The reaction mixturewas then slowly heated to 23-28° C. and stirred for 20 hours. Thereaction was then cooled to 10-15° C. and charged with 150 mL water. Themixture was stirred at 15-20° C. for 35-45 minutes and the aqueous layerwas then separated and extracted with 150 mL methylene chloride. Theorganic layers were combined and neutralized with 2.5% HCl (aq) at atemperature of 5-20° C. to give a final pH of 5-6. The organic layer wasthen washed with water and concentrated in vacuo at a temperature below20° C. to 150 mL to give compound 30 in methylene chloride.

Procedure for the Preparation of 5-nitro-2,4-di-tert-butylphenyl methylcarbonate (31)

Method 1

To a stirred solution of compound 30 (6.77 g, 25.6 mmol) was added 6 mLof a 1:1 mixture of sulfuric acid and nitric acid at 0° C. dropwise. Themixture was allowed to warm to room temperature and stirred for 1 hour.The product was purified using liquid chromatography (ISCO, 120 g, 0-7%EtOAc/Hexanes, 38 min) producing about an 8:1-10:1 mixture ofregioisomers of compound 31 as a white solid. ¹H NMR (400 MHz, DMSO-d₆)δ 7.63 (s, 1H), 7.56 (s, 1H), 3.87 (s, 3H), 1.36 (s, 9H), 1.32 (s, 9H).HPLC ret. time 3.92 min 10-99% CH₃CN, 5 min run; ESI-MS 310 m/z (MH)⁺.

Method 2

To compound 30 (100 g, 378 mmol) was added DCM (540 g, 408 mL). Themixture was stirred until all solids dissolved, and then cooled to −5-0°C. Concentrated sulfuric acid (163 g) was then added dropwise, whilemaintaining the initial temperature of the reaction, and the mixture wasstirred for 4.5 hours. Nitric acid (62 g) was then added dropwise over2-4 hours while maintaining the initial temperature of the reaction, andwas then stirred at this temperature for an additional 4.5 hours. Thereaction mixture was then slowly added to cold water, maintaining atemperature below 5° C. The quenched reaction was then heated to 25° C.and the aqueous layer was removed and extracted with methylene chloride.The combined organic layers were washed with water, dried using Na₂SO₄,and concentrated to 124-155 mL. Hexane (48 g) was added and theresulting mixture was again concentrated to 124-155 mL. More hexane (160g) was subsequently added to the mixture. The mixture was then stirredat 23-27° C. for 15.5 hours, and was then filtered. To the filter cakewas added hexane (115 g), the resulting mixture was heated to reflux andstirred for 2-2.5 hours. The mixture was then cooled to 3-7° C., stirredfor an additional 1-1.5 hours, and filtered to give compound 31 as apale yellow solid.

Procedure for the Preparation of 5-amino-2,4-di-tert-butylphenyl methylcarbonate (32)

2,4-Di-tert-butyl-5-nitrophenyl methyl carbonate (1.00 eq) was chargedto a suitable hydrogenation reactor, followed by 5% Pd/C (2.50 wt % drybasis, Johnson-Matthey Type 37). MeOH (15.0 vol) was charged to thereactor, and the system was closed. The system was purged with N₂ (g),and was then pressurized to 2.0 Bar with H₂ (g). The reaction wasperformed at a reaction temperature of 25° C.+/−5° C. When complete, thereaction was filtered, and the reactor/cake was washed with MeOH (4.00vol). The resulting filtrate was distilled under vacuum at no more than50° C. to 8.00 vol. Water (2.00 vol) was added at 45° C.+/−5° C. Theresultant slurry was cooled to 0° C.+/−5. The slurry was held at 0°C.+/−5° C. for no less than 1 hour, and filtered. The cake was washedonce with 0° C.+/−5° C. MeOH/H₂O (8:2) (2.00 vol). The cake was driedunder vacuum (−0.90 bar and −0.86 bar) at 35° C.-40° C. to give compound32. ¹H NMR (400 MHz, DMSO-d₆) δ 7.05 (s, 1H), 6.39 (s, 1H), 4.80 (s,2H), 3.82 (s, 3H), 1.33 (s, 9H), 1.23 (s, 9H).

Once the reaction was complete, the resulting mixture was diluted withfrom about 5 to 10 volumes of MeOH (e.g., from about 6 to about 9volumes of MeOH, from about 7 to about 8.5 volumes of MeOH, from about7.5 to about 8 volumes of MeOH, or about 7.7 volumes of MeOH), heated toa temperature of about 35±5° C., filtered, washed, and dried, asdescribed above.

Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 2)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P® 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to25.0° C.+/−2.5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture.The reaction mixture was washed with water (10.0 vol) 2 times. 2-MeTHFwas added to bring the total volume of reaction to 40.0 vol (˜16.5 volcharged). To this solution was added NaOMe/MeOH (1.7 equiv) to performthe methanolysis. The reaction was stirred for no less than 1.0 hour,and checked for completion by HPLC. Once complete, the reaction wasquenched with 1 N HCl (10.0 vol), and washed with 0.1 N HCl (10.0 vol).The organic solution was polish filtered to remove any particulates andplaced in a second reactor. The filtered solution was concentrated at nomore than 35° C. (jacket temperature) and no less than 8.0° C. (internalreaction temperature) under reduced pressure to 20 vol. CH₃CN was addedto 40 vol and the solution concentrated at no more than 35° C. (jackettemperature) and no less than 8.0° C. (internal reaction temperature) to20 vol. The addition of CH₃CN and concentration cycle was repeated 2more times for a total of 3 additions of CH₃CN and 4 concentrations to20 vol. After the final concentration to 20 vol, 16.0 vol of CH₃CN wasadded followed by 4.0 vol of H₂O to make a final concentration of 40 volof 10% H₂O/CH₃CN relative to the starting acid. This slurry was heatedto 78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 0.0° C.+/−5° C. over 5 hours, andfiltered. The cake was washed with 0.0° C.+/−5.0° C. CH₃CN (5 vol) 4times. The resulting solid (Compound 2) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Alternative Preparation ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 2)

4-Oxo-1,4-dihydroquinoline-3-carboxylic acid, 26, (1.0 eq) and5-amino-2,4-di-tert-butylphenyl methyl carbonate, 32, (1.1 eq) werecharged to a reactor. 2-MeTHF (4.0 vol, relative to the acid) was addedfollowed by T3P′ 50% solution in 2-MeTHF (1.7 eq). The T3P chargedvessel was washed with 2-MeTHF (0.6 vol). Pyridine (2.0 eq) was thenadded, and the resulting suspension was heated to 47.5+/−5.0° C. andheld at this temperature for 8 hours. A sample was taken and checked forcompletion by HPLC. Once complete, the resulting mixture was cooled to20° C.+/−5° C. 2-MeTHF was added (12.5 vol) to dilute the mixture. Thereaction mixture was washed with water (10.0 vol) 2 times and 2-MeTHF(16.5 vol) was charged to the reactor. This solution was charged with30% w/w NaOMe/MeOH (1.7 equiv) to perform the methanolysis. The reactionwas stirred at 25.0° C.+/−5.0° C. for no less than 1.0 hour, and checkedfor completion by HPLC. Once complete, the reaction was quenched with1.2 N HCl/H₂O (10.0 vol), and washed with 0.1 N HCl/H₂O (10.0 vol). Theorganic solution was polish filtered to remove any particulates andplaced in a second reactor.

The filtered solution was concentrated at no more than 35° C. (jackettemperature) and no less than 8.0° C. (internal reaction temperature)under reduced pressure to 20 vol. CH₃CN was added to 40 vol and thesolution concentrated at no more than 35° C. (jacket temperature) and noless than 8.0° C. (internal reaction temperature) to 20 vol. Theaddition of CH₃CN and concentration cycle was repeated 2 more times fora total of 3 additions of CH₃CN and 4 concentrations to 20 vol. Afterthe final concentration to 20 vol, 16.0 vol of CH₃CN was chargedfollowed by 4.0 vol of H₂O to make a final concentration of 40 vol of10% H₂O/CH₃CN relative to the starting acid. This slurry was heated to78.0° C.+/−5.0° C. (reflux). The slurry was then stirred for no lessthan 5 hours. The slurry was cooled to 20 to 25° C. over 5 hours, andfiltered. The cake was washed with CH₃CN (5 vol) heated to 20 to 25° C.4 times. The resulting solid (Compound 2) was dried in a vacuum oven at50.0° C.+/−5.0° C. ¹H NMR (400 MHz, DMSO-d₆) δ 12.8 (s, 1H), 11.8 (s,1H), 9.2 (s, 1H), 8.9 (s, 1H), 8.3 (s, 1H), 7.2 (s, 1H), 7.9 (t, 1H),7.8 (d, 1H), 7.5 (t, 1H), 7.1 (s, 1H), 1.4 (s, 9H), 1.4 (s, 9H).

Procedure for the Recrystallization ofN-(2,4-di-tert-butyl-5-hydroxyphenyl)-4-oxo-1,4-dihydroquinoline-3-carboxamide(Compound 2)

Compound 2 (1.0 eq) was charged to a reactor. 2-MeTHF (20.0 vol) wasadded followed by 0.1N HCl (5.0 vol). The biphasic solution was stirredand separated and the top organic phase was washed twice more with 0.1NHCl (5.0 vol). The organic solution was polish filtered to remove anyparticulates and placed in a second reactor. The filtered solution wasconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0° C. (internal reaction temperature) under reduced pressure to10 vol. Isopropyl acetate (IPAc) (10 vol) was added and the solutionconcentrated at no more than 35° C. (jacket temperature) and no morethan 8.0° C. (internal reaction temperature) to 10 vol. The addition ofIPAc and concentration was repeated 2 more times for a total of 3additions of IPAc and 4 concentrations to 10 vol. After the finalconcentration, 10 vol of IPAc was charged and the slurry was heated toreflux and maintained at this temperature for 5 hours. The slurry wascooled to 0.0° C.+/−5° C. over 5 hours and filtered. The cake was washedwith IPAc (5 vol) once. The resulting solid was dried in a vacuum ovenat 50.0° C.+/−5.0° C.

Preparation of a Solid Dispersion Comprising Substantially AmorphousCompound 2

A solvent system of MEK and DI water, formulated according to the ratio90 wt % MEK/10 wt % DI water, was heated to a temperature of 20-30° C.in a reactor, equipped with a magnetic stirrer and thermal circuit. Intothis solvent system, hypromellose acetate succinate polymer (HPMCAS)(HGgrade), SLS, and Compound 2 were added according to the ratio 19.5 wt %hypromellose acetate succinate/0.5 wt % SLS/80 wt % Compound 2. Theresulting mixture contained 10.5 wt % solids. The actual amounts ofingredients and solvents used to generate this mixture are recited inTable 5, below:

TABLE 5 Solid Spray Dispersion Ingredients for Intermediate F. UnitsBatch Compound 2 Kg 70.0 HPMCAS Kg 17.1 SLS Kg 0.438 Total Solids Kg87.5 MEK Kg 671 Water Kg 74.6 Total Solvents Kg 746 Total Spray SolutionWeight Kg 833

The mixture temperature was adjusted to a range of 20-45° C. and mixeduntil it was substantially homogenous and all components weresubstantially dissolved.

A spray drier, Niro PSD4 Commercial Spray Dryer, fitted with pressurenozzle (Spray Systems Maximum Passage series SK-MFP having orifice/coresize 54/21) equipped with anti-bearding cap, was used under normal spraydrying mode, following the dry spray process parameters recited in Table6, below.

TABLE 6 Dry spray process parameters used to generate Intermediate F.Parameter Value Feed Pressure 20 bar Feed Flow Rate 92-100 Kg/hr InletTemperature 93-99° C. Outlet Temperature 53-57° C. Vacuum DryerTemperature 80° C. for 2 hours then 110° C. (+/−5° C.) Vacuum DryingTime 20-24 hours

A high efficiency cyclone separated the wet product from the spray gasand solvent vapors. The wet product contained 8.5-9.7% MEK and0.56-0.83% Water and had a mean particle size of 17-19 um and a bulkdensity of 0.27-0.33 g/cc. The wet product was transferred to a 4000Lstainless steel double cone vacuum dryer for drying to reduce residualsolvents to a level of less than about 5000 ppm and to generate dryspray dry dispersion of amorphous Compound 2, containing <0.03% MEK and0.3% Water.

Tablet Formation from a Fully Continuous Wet Granulation Process

Equipment/Process

Equipment

Fully Continuous Development and Launch Rig (DLR) or similar type ofequipment.

Screening

Compound 1 Form I, the solid dispersion comprising substantiallyamorphous Compound 2, and excipients may be dispensed in separateintermediate bin containers (IBCs). These materials may be screenedusing a “bin-to-bin” screening operation. Appropriate screen sizes aremesh 20, mesh 40, or mesh 60.

Blending

The IBCs containing the screened Compound 1 Form I, the solid dispersioncomprising substantially amorphous Compound 2, and excipients may bedocked to the a feeder system, which can feed the materials in acontrolled manner, e.g. using volumetric or gravimetric loss in weightfeeders, into a continuous blender. The feed rates of the individualcomponents is defined by the formulation composition and the overallline rate. The line rate may be 8 kg/hr to 30 kg/hr. The continuousblender can have different blade configurations to allow appropriateblending and the rotational speed of these blades may be between 80 RPMand 300 RPM.

Wet Granulation

A granulation solution may be prepared by dissolving 48 g sodium laurylsulfate and 159 g polyvinylpyrrolidone in 1,626 g water in a stainlesssteel container, using an overhead stirrer with a stirring speed of 700RPM. The granulation solution may be placed in a container from whichthe solution may be pumped into the twin screw granulator using aperistaltic pump with a mass flow meter and control, using a flow ratethat is appropriate for the process. The blend may be granulated using atwin screw granulator such as the granulator that is part of the DLR.The blend may be added to the twin screw granulator using a Loss inWeight feeder, such as the K-Tron feeder on the DLR, with a feed rate of8 kg/hr to 24 kg/hr. The twin screw granulator may be operated with abarrel temperature of 25 degrees Celsius and a screw speed of 200 to 950RPM. The granulation process may be performed for three minutes forsmall batch sizes or several hours for large batch sizes.

Drying

The wet granules may be fed directly into a fluid bed dryer, such as thesegmented fluid bed dryer on the DLR. The drying end-point may be chosenat a product temperature during discharge ranging from 40 to 55 degreesCelsius at which point the water content of the granules may be 2.1% w/w(“Loss on Drying, LOD”) or less. The drying time may be 12 minutes, orshorter or longer, to reach the desired drying endpoint.

Milling

The dried granules may be milled to reduce the size of the granules. Acone mill such as the integrated Quadro U 10 CoMil may be used for this.

Blending

The granules may be blended with extra-granular excipients such asfillers and lubricant using loss in weight feeders and a continuousblender. The blending speed may be 80-300 RPM.

Compression

The compression blend may be compressed into tablets using a singlestation or rotary tablet press, such as the Courtoy Modul P press, whichis part of the DLR system, using appropriately sized tooling. The weightof the tablets for a dose of 200 mg of Compound 1 Form I and 125 mg ofsubstantially amorphous Compound 2 may be about 500 or 600 mg.

Film Coating

Tablets may be film coated using the innovative Omega film coater, whichis part of the DLR system. This coater enables fast film coating ofsub-batches of 1 to 4 kg to allow continuous manufacturing.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, an Ackley ramp printer.

The continuous process described above in one embodiment is enhanced byPAT techniques as described in Table 7. There are 6 PAT positions eachof which includes a manual sampling port. In process samples can beobtained for investigational reasons, as needed, and also for PAT modelmaintenance, transfer, and validation. The PAT systems may be used forreal time release testing (RTRT) and may also be employed for in processcontrols (IPC) and feedback/feed-forward control.

TABLE 7 Processing Proposed Location Technology Step Purpose Role PAT 1NIR Dispensing/ Build an NIR IPC Charging raw material library PAT 2 NIRInitial blend Blend IPC uniformity PAT 3 NIR Wet Granulation Granule IPCuniformity RTRT/IPC Moisture Laser Wet Granulation Particle size RTRTDiffraction distribution PAT 4 NIR Final blend Blend RTRT uniformityRTRT Moisture PAT 5 Raman Compression API form RTRT Identification RTRTTablet Compression Weight RTRT/IPC Tester Thickness IPC HardnessRTRT/IPC PAT 6 Raman Coating Coating IPC thickness

Meeting specifications may be done by RTRT as described in Table 8.

TABLE 8 Final Product In-Process Attribute PAT Position MaterialMeasurement Identity PAT 5a (Raman) Uncoated Tablet Confirms spectrummatches the reference standard spectra Assay PAT 4 (NIR) Final Blend APIConcentration PAT 5b (Tablet Uncoated Tablet Tablet Weight Tester) CUPAT 4 (NIR) Final Blend Variance in API concentration PAT 5b (TabletUncoated Tablet Variance in Tester) tablet weight Dissolution Mayinclude: May include: PAT 3b (Laser Milled granules Granule ParticleDiffraction) Size PAT 4 (NIR) Final Blend API Concentration PAT 5b(Tablet Uncoated Tablet Tablet Weight, Tester) Hardness Moisture PAT 4Final Blend Water Content Form PAT 5a (Raman) Uncoated Tablet Form I &Absence of Form II

There is a high probability of detecting non-conforming material. Forexample, if model classification criterion is set at a minimum of 95%confidence and 800 tablets are tested during batch manufacture, 40 hourrun with a sampling rate of 1 tablet every 3 minutes equals 800 tablets.Then, probability of passing a non-conforming batch is extremely low:<(0.05)^(n-), where n=# of samples, therefore the probability is<1.5×10⁻¹⁰⁴¹. Probability of not detecting non-conforming tabletsresulting from a short term event (≥3 minutes) is as follows: 1 tablet(3 min event)→0.05 (probability of detection >0.95); 2 tablets (6 minuteevent)→0.0025 (probability of detection >0.9975).

PAT measurements can serve as surrogates for conventional end-testingdirectly via combining measurements to express attributes conventionally(i.e. as assay, CU, dissolution, etc.). Validation can be performedusing ICH Q2 as guidance. Sequential off-line to on-line methoddevelopment allows for the assessment of CQAs in a material sparingmanner. Ultimately, RTRT will lead to ensuring product quality at ahigher confidence level than conventional testing.

Tablet Formation from Twin Screw Wet Granulation Process

Equipment/Process

Equipment

Twin Screw Wet Granulators: ConsiGma-1, ConsiGma-25 or Leistritz nano.

Screening/Weighing

Compound 1 Form I, the solid dispersion comprising substantiallyamorphous Compound 2, and excipients may be screened prior to or afterweigh-out. Appropriate screen sizes are mesh 20, mesh 40, or mesh 60.Compound 1 Form I and/or the solid dispersion comprising substantiallyamorphous Compound 2 may be pre-blended with one or more of theexcipients to simplify screening.

Blending

Compound 1 Form I, the solid dispersion comprising substantiallyamorphous Compound 2, and excipients may be added to the blender indifferent order. The blending may be performed in a Turbula blender, av-shell blender, or a bin blender. The components may be blended for 10minutes.

Wet Granulation

A granulation solution may be prepared by dissolving 48 g sodium laurylsulfate and 159 g polyvinylpyrrolidone in 1,626 g water in a stainlesssteel container, using an overhead stirrer with a stirring speed of 700RPM. The blend may be granulated using a twin screw granulator such asthe ConsiGma-1. The granulation solution may be added to the twin screwgranulator using a peristaltic pump, such as the pump on the ConsiGma-1,with a feed rate of 67 g/min. The blend may be added to the twin screwgranulator using a Loss in Weight feeder, such as the Brabender feederon the ConsiGma-1, with a feed rate of 10 kg/hr. The twin screwgranulator may be operated with a barrel temperature of 25 degreesCelsius and a screw speed of 400 RPM. The granulation process may beperformed for four minutes. The granulation process may be performed fora shorter or longer duration of time to produce a smaller or largeramount of wet granules.

Drying

The wet granules may be fed directly into a fluid bed dryer, such as thedrying chamber on the ConsiGma-1 or the segmented fluid bed dryer on theCTL-25. The drying end-point may be chosen at a product temperature of43 degrees Celsius at which point the water content of the granules maybe 1.6% w/w (“Loss on Drying, LOD”). The drying time may be 12 minutes,or shorter or longer, to reach the desired drying endpoint. The dryingmay be performed with an air flow of 59 m³/min and inlet temperature of60 degrees Celsius. Alternatively, the wet granules coming from the twinscrew granulator may be collected into a bin or container for a certainperiod of time after which the wet granules are transferred to aseparate stand-alone fluid bed dryer, such as the Vector Multi 15.

Milling

The dried granules may be milled to reduce the size of the granules. Acone mill such as the Quadro 194 CoMil may be used for this.

Blending

The granules may be blended with extra-granular excipients such asfillers and lubricant using a V-shell blender or a bin blender. Theblending time may be 5, 3 or 1 minute(s).

Compression

The compression blend may be compressed into tablets using a singlestation or rotary tablet press, such as the Courtoy Modul P press, using0.55′×0.33′ oval shaped tooling. The weight of the tablets for a dose of200 mg of Compound 1 Form I and 125 mg of substantially amorphousCompound 2 may be about 500 or 600 mg.

Film Coating

Tablets may be film coated using a pan coater, such as, for example aThomas Engineering Compu-Lab coater. A trace amount of Carnauba wax maybe added to improve tablet appearance and process ability.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, a Hartnett Delta printer.

Tablet Formation from Continuous Twin Screw Wet Granulation Process

Equipment/Process

Equipment Granulator: ConsiGma or Leistritz or Thermo Fisher twin screwgranulator.

Screening/Weighing

Compound 1 and excipients may be screened prior to or after weigh-out.Possible screen sizes are mesh 20, mesh 40, or mesh 60. Compound 1 maybe pre-blended with one or more of the excipients to simplify screening.

Blending

Compound 1 and excipients may be added to the blender in differentorder. The blending may be performed in a Turbula blender, a v-shellblender, a bin blender, or a continuous blender. The components may beblended for 10 minutes for batch blenders or continuously for acontinuous blender.

Granulation Operation

Granulation Fluid—SLS and binder are added to purified water and mixeduntil dissolved. A suitable ratio is 2.5% w/w SLS and 10.0% w/w PVP K30in water.

Granulation—The blend containing Compound 1 and excipients may be dosedinto the twin screw granulator using a Loss in Weight feeder at a rateof 10 kg/hr. The granulation fluid may be added using a peristaltic pumpat a rate of 3.5 kg/hr. The granulator may be run at a speed of 400 RPM.A notable advantage of the present twin screw wet granulation process isusing a granulation fluid that comprises both a surfactant and thebinder for better granulation through increased wettability. In oneembodiment, the surfactant is SLS. Another notable advantage is thatbecause the process is continuous and at any moment in time only alimited amount of material is processed, the process can be wellcontrolled and results in a high quality product.

Milling

The granules may be reduced in size using a screen mill or a cone mill,either before drying or after drying, or both.

Drying

The granules may be dried using a vacuum oven, tray dryer, bi-conicaldryer, or fluid bed drier.

Blending

The granules may be blended with extra-granular excipients. The granuleshave been blended using a 300 liter bin blender for 60 revolutions.

Compression

The compression blend has been compressed into tablets using a CourtoyModul P rotary press

Film Coating

Tablets may be film coated using a pan coater, such as, for example anO'Hara Labcoat.

Printing

Film coated tablets may be printed with a monogram on one or both tabletfaces with, for example, a Hartnett Delta printer.

Assays

Protocol 1

Assays for Detecting and Measuring ΔF508-CFTR Potentiation Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluorescent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-free bath solution: Chloride salts in Bath Solution #1 (above)are substituted with gluconate salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, P-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours.

Electrophysiological Assays for assaying ΔF508-CFTR modulationproperties of compounds.

Ussing Chamber Assay

Ussing chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRaugmenters or inducers identified in the optical assays. Non-CF and CFairway epithelia were isolated from bronchial tissue, cultured aspreviously described (Galietta, L. J. V., Lantero, S., Gazzolo, A.,Sacco, O., Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In VitroCell. Dev. Biol. 34, 478-481), and plated onto Costar® Snapwell™ filtersthat were precoated with NIH3T3-conditioned media. After four days theapical media was removed and the cells were grown at an air liquidinterface for >14 days prior to use. This resulted in a monolayer offully differentiated columnar cells that were ciliated, features thatare characteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUsing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical Cl⁻ gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,Iowa). Briefly, HBE were examined under voltage-clamp recordingconditions (V_(hold)=0 mV) at 37° C. The basolateral solution contained(in mM) 145 NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apicalsolution contained (in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

Patch-Clamp Recordings

Total Cl⁻ current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mLamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≤2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Protocol 2

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane potential optical methods for assaying ΔF508-CFTR modulationproperties of compounds.

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See. Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 M forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

Solutions

-   -   Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1,        HEPES 10, pH 7.4 with NaOH.    -   Chloride-free bath solution: Chloride salts in Bath Solution #1        (above) are substituted with gluconate salts.    -   CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored        at −20° C.    -   DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20°        C.        Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, 13-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

Electrophysiological Assays for assaying ΔF508-CFTR modulationproperties of compounds

Ussing Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR augmentersor inducers identified in the optical assays. FRT^(ΔF508-CFTR)epithelial cells grown on Costar Snapwell cell culture inserts weremounted in an Ussing chamber (Physiologic Instruments, Inc., San Diego,Calif.), and the monolayers were continuously short-circuited using aVoltage-clamp System (Department of Bioengineering, University of Iowa,Iowa, and, Physiologic Instruments, Inc., San Diego, Calif.).Transepithelial resistance was measured by applying a 2-mV pulse. Underthese conditions, the FRT epithelia demonstrated resistances of 4 KΩ/cm²or more. The solutions were maintained at 27° C. and bubbled with air.The electrode offset potential and fluid resistance were corrected usinga cell-free insert. Under these conditions, the current reflects theflow of Cl⁻ through ΔF508-CFTR expressed in the apical membrane. TheI_(SC) was digitally acquired using an MP100A-CE interface andAcqKnowledge software (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

Solutions

-   -   Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂        (1.2), K₂HPO₄ (2.4), KHPO₄ (0.6),        N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES)        (10), and dextrose (10). The solution was titrated to pH 7.4        with NaOH.    -   Apical solution (in mM): Same as basolateral solution with NaCl        replaced with Na Gluconate (135).        Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR augmenters or inducers identified from our opticalassays. The cells were cultured on Costar Snapwell cell culture insertsand cultured for five days at 37° C. and 5% CO₂ in Coon's modified Ham'sF-12 medium supplemented with 5% fetal calf serum, 100 U/ml penicillin,and 100 μg/ml streptomycin. Prior to use for characterizing thepotentiator activity of compounds, the cells were incubated at 27° C.for 16-48 hrs to correct for the ΔF508-CFTR. To determine the activityof corrections compounds, the cells were incubated at 27° C. or 37° C.with and without the compounds for 24 hours.

Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 G1 and a series resistance <15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 M of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Solutions

-   -   Intracellular solution (in mM): Cs-aspartate (90), CsCl (50),        MgCl₂ (1), HEPES (10), and 240 g/ml amphotericin-B (pH adjusted        to 7.35 with CsOH).    -   Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl        (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35        with HCl).        Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F⁻ (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(p)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing ≤2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Solutions

-   -   Extracellular solution (in mM): NMDG (150), aspartic acid (150),        CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with        Tris base).    -   Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA        (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with        HCl).        Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compound 1 and Compound 2 of the invention are useful as augmenters orinducers of CFTR activity. Table 9 below illustrates the EC50 andrelative efficacy of Compound 1 and Compound 2. In Table 9 below, thefollowing meanings apply. EC50: “+++” means <10 uM; “++” means between10 uM to 25 uM; “+” means between 25 uM to 60 uM. % Efficacy: “+” means<25%; “++” means between 25% to 100%; “+++” means >100%.

TABLE 9 Cmpd. No. EC50 (μM) % Activity 1 +++ +++ 2 +++ ++

Other Embodiments

All publications and patents referred to in this disclosure areincorporated herein by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference. Should themeaning of the terms in any of the patents or publications incorporatedby reference conflict with the meaning of the terms used in thisdisclosure, the meaning of the terms in this disclosure are intended tobe controlling. Furthermore, the foregoing discussion discloses anddescribes merely exemplary embodiments of the invention. One skilled inthe art will readily recognize from such discussion and from theaccompanying drawings and claims, that various changes, modificationsand variations can be made therein without departing from the spirit andscope of the invention as defined in the following claims.

The invention claimed is:
 1. A continuous process for preparing a tabletcomprising3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid (“Compound 1”) Form I and a solid dispersion comprisingsubstantially amorphousN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide(“Compound 2”) comprising the steps of: a) mixing Compound 1 Form I, asolid dispersion comprising substantially amorphous Compound 2, afiller, and a disintegrant in a blender to form a blend; b) preparing agranulation solution with water, a binder, and a surfactant; c) feedingthe blend from step a) into a continuous twin screw granulator whileadding the granulation solution from step b) to produce granules; d)drying the granules from step c) and milling them; e) blending themilled granules from step d) with a filler, disintegrant, and lubricantto form a blend; and f) compressing the blend from step e) into atablet; wherein at least one of the above steps comprises processanalytical technology; and wherein step f) comprises monitoring solid,form identity of Compound 1 and/or Compound 2 in the tablet using Ramanspectroscopy.
 2. The process of claim 1, wherein the process analytictechnology comprises NIR and/or laser diffraction to monitor definedstandards.
 3. The process of claim 2, wherein the defined standard isselected from blend uniformity, granule uniformity, moisture, particlesize distribution, active pharmaceutical ingredient solid form identity,weight, thickness, hardness, and coating thickness.
 4. The process ofclaim 1, wherein step a) comprises monitoring blend uniformity usingNIR.
 5. The process of claim 1, wherein step c) comprises monitoringgranule uniformity and/or moisture using NIR.
 6. The process of claim 1,wherein step c) comprises monitoring particle size distribution usinglaser diffraction.
 7. The process of claim 1, wherein step e) comprisesmonitoring blend uniformity and/or moisture using NIR.
 8. The process ofclaim 1, wherein step f) further comprises monitoring tablet weight,thickness, and/or hardness using a tablet tester.